Methods for diagnosing and treating inflammatory bowel disease

ABSTRACT

Biomarkers predictive of responsiveness to integrin beta7 antagonists, including anti-beta7 integrin subunit antibodies, and methods of using such biomarkers are provided. In addition, methods of treating gastrointestinal inflammatory disorders such as inflammatory bowel diseases including ulcerative colitis and Crohn&#39;s disease are provided. Also provided are methods of using such predictive biomarkers for the treatment of inflammatory bowel diseases including ulcerative colitis and Crohn&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2013/063384 having an international filing date of Oct. 4, 2013,which claims the benefit of priority of provisional U.S. Application No.61/860,422 filed Jul. 31, 2013 and provisional U.S. Application No.61/710,656 filed Oct. 5, 2012, all of which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 23, 2013, isnamed P4989R1WO_SL.txt and is 19,125 bytes in size.

FIELD

Biomarkers predictive of responsiveness to integrin beta7 antagonists,including anti-beta7 integrin subunit antibodies, and methods of usingsuch biomarkers are provided. In addition, methods of treatinggastrointestinal inflammatory disorders such as inflammatory boweldiseases including ulcerative colitis and Crohn's disease are provided.Also provided are methods of using such predictive biomarkers for thetreatment of inflammatory bowel diseases including ulcerative colitisand Crohn's disease.

BACKGROUND

Inflammatory bowel disease (IBD) is a chronic inflammatory autoimmunecondition of the gastrointestinal (GI) tract, which presents clinicallyas either ulcerative colitis (UC) or Crohn's disease (CD). CD is achronic transmural inflammatory disease with the potential to affect anypart of the entire GI tract, and UC is a mucosal inflammation of thecolon. Both conditions are characterized clinically by frequent bowelmotions, malnutrition, and dehydration, with disruption in theactivities of daily living. CD is frequently complicated by thedevelopment of malabsorption, strictures, and fistulae and may requirerepeated surgery. UC, less frequently, may be complicated by severebloody diarrhea and toxic megacolon, also requiring surgery. Both IBDconditions are associated with an increased risk for malignancy of theGI tract. The etiology of IBD is complex, and many aspects of thepathogenesis remain unclear.

The treatment of moderate to severe IBD poses significant challenges totreating physicians, because conventional therapy with corticosteroidsand immunomodulator therapy (e.g., azathioprine, 6 mercaptopurine, andmethotrexate) is associated with side effects and intolerance and hasnot shown proven benefit in maintenance therapy (steroids). Monoclonalantibodies targeting tumor necrosis factor alpha (TNF-α), such asinfliximab (a chimeric antibody) and adalimumab (a fully humanantibody), are currently used in the management of CD. Infliximab hasalso shown efficacy and has been approved for use in UC. However,approximately 10%-20% of patients with CD are primary nonresponders toanti TNF therapy, and another ˜20%-30% of CD patients lose response overtime (Schnitzler et al., Gut 58:492-500 (2009)). Other adverse events(AEs) associated with anti TNFs include elevated rates of bacterialinfection, including tuberculosis, and, more rarely, lymphoma anddemyelination (Chang et al., Nat Clin Pract Gastroenterol Hepatology3:220 (2006); Hoentjen et al., World J. Gastroenterol. 15(17):2067(2009)). No currently available therapy achieves sustained remission inmore than 20%-30% of IBD patients with chronic disease (Hanauer et al.,Lancet 359:1541-49 (2002); Sandborn et al., N Engl J Med 353:1912-25(2005)). In addition, most patients do not achieve sustainedsteroid-free remission and mucosal healing, clinical outcomes thatcorrelate with true disease modification. Therefore, there is a need todevelop more targeted therapy in IBD that is optimized for chronic use:an improved safety profile with sustained remission, particularlysteroid-free remission and prevention of long-term complications in agreater proportion of patients, including those patients who eithernever respond to an anti TNF therapeutic agent or lose response overtime (TNF inadequate responder patients or TNF-IR patients).

The integrins are alpha/beta heterodimeric cell surface glycoproteinreceptors that play a role in numerous cellular processes includingleukocyte adhesion, signaling, proliferation, and migration, as well asin gene regulation (Hynes, R. O., Cell, 1992, 69:11-25; and Hemler, M.E., Annu. Rev. Immunol., 1990, 8:365-368). They are composed of twoheterodimeric, non-covalently interacting α and β transmembrane subunitsthat bind specifically to distinct cell adhesion molecules (CAMs) onendothelia, epithelia, and extracellular matrix proteins. In thismanner, integrins can function as tissue-specific cell adhesionreceptors aiding in the recruitment of leukocytes from blood into nearlyall tissue sites in a highly regulated manner, playing a role in thehoming of leukocytes to normal tissue and to sites of inflammation (vonAndrian et al., N Engl J Med 343:1020-34 (2000)). In the immune system,integrins are involved in leukocyte trafficking, adhesion andinfiltration during inflammatory processes (Nakajima, H. et al., J. Exp.Med., 1994, 179:1145-1154). Differential expression of integrinsregulates the adhesive properties of cells and different integrins areinvolved in different inflammatory responses. (Butcher, E. C. et al.,Science, 1996, 272:60-66). The beta7 containing integrins (i.e.,alpha4beta7 and alphaEbeta7) are expressed primarily on monocytes,lymphocytes, eosinophils, basophils, and macrophages but not onneutrophils (Elices, M. J. et al., Cell, 1990, 60:577-584).

The α4β7 integrin is a leukocyte-homing receptor that is important inthe migration of cells to the intestinal mucosa and associated lymphoidtissues, such as Peyer's patches in the small intestine, lymphoidfollicles in the large intestine, and mesenteric lymph nodes. In thegut, leukocyte rolling and firm adhesion to the mucosal endothelium isinitiated by signals from chemokines and is mediated via mucosaladdressin cell adhesion molecule (MAdCAM)-1-associated sialyl Lewis X.Chemokine signaling induces the α4β7 integrin to undergo a change fromlow to high MAdCAM-1 binding affinity. The leukocyte then arrests andbegins the process of extravasation through the vascular endothelium tounderlying tissue. This extravasation process is believed to occur inboth the normal immune cell recirculation state and in inflammatoryconditions (von Andrian et al., supra). The numbers of α4β7⁺ cells ininfiltrates and the expression of the ligand MAdCAM-1 are higher atsites of chronic inflammation such as in the intestinal tract ofpatients with UC or CD (Briskin et al., Am J Pathol 151:97-110 (1997);Souza et al., Gut 45:856-63 (1999)). α4β7 binds preferentially to highendothelial venules expressing MAdCAM-1 and vascular cell adhesionmolecule (VCAM)-1, as well as to the extracellular matrix moleculefibronectin fragment CS-1 (Chan et al., J Biol Chem 267:8366-70 (1992);Ruegg et al., J Cell Biol 17:179-89 (1992); Berlin et al., Cell74:185-95 (1993)). Together with constitutively expressed MAdCAM-1 ingut mucosal vessels, the α4β7 integrin plays a selective role inleukocyte gut tropism but does not seem to contribute to homing ofleukocytes to the peripheral tissue or the CNS. Instead, peripherallymphoid trafficking has been associated with α4β1 interaction withVCAM-1 (Yednock et al., Nature 356:63-6 (1992); Rice et al., Neurology64:1336-42 (2005)).

Another member of the β7 integrin family, expressed exclusively on Tlymphocytes and associated with mucosal tissues, is the αEβ7 integrin,otherwise known as CD103. The αEβ7 integrin binds selectively toE-cadherin on epithelial cells and has been proposed to play a role inthe retention of T cells in the mucosal tissue in the intraepitheliallymphocyte compartment (Cepek et al., J Immunol 150:3459-70 (1993);Karecla et al. Eur J Immunol 25:852-6 (1995)). The αEβ7⁺ cells in thelamina propria have been reported to exhibit cytotoxicity againststressed or infected epithelial cells (Hadley et al., J Immunol159:3748-56 (1997); Buri et al., J Pathol 206:178-85 (2005)). Theexpression of αEβ7 is increased in CD (Elewaut et al., ActaGastroenterol Belg 61:288-94 (1998); Oshitani et al., Int J Mol Med12:715-9 (2003)), and anti-αEβ7 antibody treatment has been reported toattenuate experimental colitis in mice, implicating a role for αEβ7⁺lymphocytes in experimental models of IBD (Ludviksson et al., J Immunol162:4975-82 (1999)).

Administration of monoclonal antibodies against alphaE beta7 reportedlyprevents and ameliorates immunization induced colitis in IL-2^(−/−)mice, suggesting that the onset and maintenance of inflammatory boweldisease depends on colonic localization of lamina propria CD4lymphocytes expressing alphaEbeta7 (Ludviksson et al., J Immunol. 1999,162(8):4975-82). An anti-α4 antibody (natalizumab) reportedly hasefficacy in treatment of patients with CD (Sandborn et al., N Engl J Med2005; 353:1912-25) and an anti-α4β7 antibody (MLN-02, MLN0002,vedolizumab) reportedly is effective in patients with UC (Feagan et al.,N Engl J Med 2005; 352:2499-507). A second anti-alpha4/beta7 antibody(AMG 181) is also in development and clinical trials have recently begun(clinicaltrials(dot)gov identifier, NCT01164904, September 2012). Thesestudies and findings validate α4β7 as a therapeutic target and supportthe idea that the interaction between α4β7 and MAdCAM-1 mediates thepathogenesis of IBD. Thus, antagonists of beta7 integrin are of greatpotential as a therapeutic agent in treating IBD.

Humanized monoclonal antibodies targeted against the β7 integrin subunithave been described previously. See, e.g., Intn'l Patent Pub. No.WO2006/026759. One such antibody, rhuMAb Beta7 (etrolizumab) is derivedfrom the rat anti-mouse/human monoclonal antibody FIB504 (Andrew et al.1994). It was engineered to include human IgG1-heavy chain and κ1-lightchain frameworks. Intn'l Patent Pub. No. WO2006/026759. Administrationof etrolizumab to human patients according to certain dosing regimenshas been described previously. See, e.g., Intn'l Patent Pub. No.WO/2012/135589.

RhuMAb Beta7 (etrolizumab) binds α4β7 (Holzmann et al., Cell 56:37-46(1989); Hu et al., Proc Natl Acad Sci USA 89:8254-8 (1992)) and αEβ7(Cepek et al., J Immunol 150:3459-70 (1993)), which regulate traffickingand retention of lymphocyte subsets, respectively, in the intestinalmucosa. Clinical studies have demonstrated the efficacy of an anti α4antibody (natalizumab) for the treatment of CD (Sandborn et al., N EnglJ Med 353:1912-25 (2005)), and encouraging results have been reportedfor anti α4β7 antibody (LDP02/MLN02/MLN0002/vedolizumab) in thetreatment of UC (Feagan et al., N Engl J Med 352:2499-507 (2005), Feaganet al., N Engl J Med 369(8):699-710 (2013)) and also CD (Sandborn etal., N Engl J Med 369(8):711-721 (2013)) These findings help to validateα4β7 as a potential therapeutic target and support the hypothesis thatthe interaction between α4β7 and mucosal addressin cell adhesionmolecule 1 (MAdCAM 1) contributes to the pathogenesis of inflammatorybowel disease (IBD).

Unlike natalizumab, which binds α4 and thus binds both α4β1 and α4β7,rhuMAb Beta7 binds specifically to the β7 subunit of α4β7 and αEβ7 anddoes not bind to α4 or β1 integrin individual subunits. This wasdemonstrated by the inability of the antibody to inhibit adhesion ofα4β1+α4β7—Ramos cells to vascular cell adhesion molecule 1 (VCAM 1) atconcentrations as high as 100 nM. Importantly, this characteristic ofrhuMAb Beta7 indicates selectivity: T cell subsets expressing α4β1 butnot β7 should not be directly affected by rhuMAb Beta7.

Support for the gut-specific effects of rhuMAb Beta7 on leukocyte homingcomes from several in vivo nonclinical studies. In severe combinedimmunodeficient (SCID) mice reconstituted with CD45RB^(high)CD4+ T cells(an animal model of colitis), rhuMAb Beta7 blocked radiolabeledlymphocyte homing to the inflamed colon but did not block homing to thespleen, a peripheral lymphoid organ. See, e.g., Intn'l Patent Pub. No.WO2006/026759. In addition, the rat-mouse chimeric anti-murine β7 (antiβ7, muFIB504) was unable to reduce the histologic degree of centralnervous system (CNS) inflammation or improve disease survival in myelinbasic protein T cell receptor (MBP-TCR) transgenic mice withexperimental autoimmune encephalitis (EAE), an animal model of multiplesclerosis. Id. Furthermore, in two safety studies in cynomolgus monkeys,rhuMAb Beta7 induced a moderate increase in peripheral blood lymphocytenumbers that was largely due to a marked (approximately three- tosixfold) increase in CD45RA⁻β7^(high) peripheral blood T cells, a subsetthat is phenotypically similar to gut-homing memory/effector T cells inhumans. See, e.g., Intn'l Patent Pub. No. WO2009/140684; Stefanich etal., Br. J. Pharmacol. 162:1855-1870 (2011). In contrast, rhuMAb Beta7had minimal to no effect on the number of CD45RA+β7 intermediateperipheral blood T cells, a subset that is phenotypically similar tonaïve T cells in humans, and no effect on the number of CD45RA⁻β7^(low)peripheral blood T cells, a subset that is phenotypically similar toperipheral homing memory/effector T cells in humans, confirming thespecificity of rhuMAb Beta7 for the gut homing lymphocyte subpopulation.Intn'l Patent Pub. No. WO2009/140684; Stefanich et al., Br. J.Pharmacol. 162:1855-1870 (2011).

While clinical studies have demonstrated the efficacy of an anti-α4antibody (natalizumab) for the treatment of CD (Sandborn et al., N EnglJ Med 353:1912-25 (2005)), and encouraging results have been reportedfor anti-α4β7 antibody (LDP02/MLN02/MLN0002/vedolizumab) in thetreatment of UC, there remains a need for further improvements in thetreatment of these disorders. For example, natalizumab treatment hasbeen associated with confirmed cases of progressive multifocalleukoencephalopathy (PML) in patients with Crohn's disease (andseparately, multiple sclerosis) who received concomitant treatment withnatalizumab and immunosupressives. PML is a potentially fatalneurological condition linked to reactivation of a polyomavirus (JCvirus) and active viral replication in the brain. No known interventionscan reliably prevent PML or adequately treat PML, if it occurs. Onelimitation of vedolizumab treatment is that it is administeredintravenously which can be inconvenient for the patient and can also beassociated with undesirable or adverse events, e.g., infusion sitereactions. Accordingly, there is a need for improved therapeuticapproaches to the treatment of gastrointestinal inflammatory disorderssuch as IBD, e.g., ulcerative colitis and Crohn's disease, as well asmore desirable dosing regimens.

It is often unknown, prior to treatment, whether a patient will respondto a particular therapeutic agent or class of therapeutic agents.Accordingly, as IBD patients in general, and UC and CD patients inparticular, seek treatment, there is considerable trial and errorinvolved in the search for therapeutic agent(s) effective for aparticular patient. Such trial and error often involves considerablerisk and discomfort the patient in order to find the most effectivetherapy. Thus, there is a need for more effective means for determiningwhich patients will respond to which treatment and for incorporatingsuch determinations into more effective treatment regimens for IBDpatients.

It would therefore be highly advantageous to have additional diagnosticmethods, including predictive diagnostic methods, that can be used toobjectively identify patients most likely to respond to treatment withvarious IBD therapeutic agents, including anti-beta7 integrin subunitantibodies. Thus, there is a continuing need to identify new biomarkersassociated with ulcerative colitis, Crohn's disease as well as otherinflammatory bowel disorders and that are predictive of response toanti-beta7 integrin subunit antibodies. In addition, statistically andbiologically significant and reproducible information regarding suchassociations could be utilized as an integral component in efforts toidentify specific subsets of UC or CD patients, such as TNF-IR patients,who would be expected to significantly benefit from treatment withanti-beta7 integrin subunit antibodies, for example where thetherapeutic agent is or has been shown in clinical studies to be oftherapeutic benefit in such specific UC or CD patient subpopulation.

The invention described herein meets certain of the above-describedneeds and provides other benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety for anypurpose.

SUMMARY

The methods of the invention are based, at least in part, on thediscovery that mRNA expression levels of certain genes in intestinalbiopsies and in peripheral whole blood, as well as cellular proteinexpression levels of certain genes in intestinal biopsies are predictiveof responsiveness of patients suffering from a gastrointestinalinflammatory disorder to treatment with integrin beta7 antagonists.

Accordingly, in one aspect, methods of predicting the response of apatient suffering from a gastrointestinal inflammatory disorder to atherapy comprising an integrin beta7 antagonist are provided. In certainembodiments, a biological sample is obtained from the patient and levelsof mRNA expression are measured. In one embodiment, expression ofintegrin beta7 mRNA is measured. In one embodiment, expression ofintegrin alphaE mRNA is measured. In one embodiment, expression ofCD3epsilon is measured. In one embodiment, expression of a combinationof two or three mRNAs selected from integrin beta7, integrin alphaE, andCD3epsilon is measured. In one embodiment, the biological sample is atissue biopsy sample. In one embodiment, the biopsy is obtained fromintestinal tissue. In one embodiment, the biological sample isperipheral whole blood. In one embodiment, the peripheral whole blood iscollected in a PAXgene tube. In certain embodiments, the mRNA expressionlevel is measured by a PCR method. In one embodiment, the PCR method isqPCR. In certain embodiments, the mRNA expression level is compared to amedian value. In one embodiment, the patient is predicted to respond totherapy comprising the integrin beta7 antagonist if the mRNA expressionlevel of one or more of integrin beta7, integrin alphaE, or CD3epsilonis elevated compared to a reference value, which in certain embodimentsis a median value. In one embodiment, the patient is predicted to notrespond to therapy comprising the integrin beta7 antagonist if the mRNAexpression level of one or more of integrin beta7, integrin alphaE, orCD3epsilon is low compared to a reference value, which in certainembodiments, is a median value. In one embodiment, the response isclinical remission. In one embodiment, the response is mucosal healing.In one embodiment, the response is clinical response. In certainembodiments, remission in the patient is determined to be induced whenthe absolute Mayo Clinic Score ≤2 and no individual subscore >1, whichis also referred to as clinical remission. In certain embodiments,mucosal healing is determined to have occurred when the patient isdetermined to have an endoscopy subscore of 0 or 1 as assessed byflexible sigmoidoscopy. In certain such embodiments, patients whoexperience mucosal healing are determined to have an endoscopy subscoreof 0.

In another aspect, methods of predicting responsiveness of agastrointestinal inflammatory disorder patient to an integrin beta7antagonist treatment are provided. In one embodiment, the mRNAexpression level of integrin beta7 in a biological sample from thepatient is determined. In one embodiment, the mRNA expression level ofintegrin alphaE in a biological sample from the patient is determined.In one embodiment, the mRNA expression level of CD3epsilon in abiological sample from the patient is determined. In one embodiment, themRNA expression level of two or three mRNAs selected from integrinbeta7, integrin alphaE, and CD3epsilon in a biological sample from thepatient is determined. In certain embodiments, elevated integrin beta7mRNA expression compared to a reference value, for example, a medianvalue, or elevated integrin alphaE mRNA expression compared to areference value, for example, a median value, or elevated CD3epsilonmRNA expression compared to a reference value, for example, a medianvalue, detected in the biological sample identifies the patient as onewho is likely to respond to integrin beta7 antagonist treatment.

In yet another aspect, a biological sample is obtained from the patientand the number of cells expressing one or a combination of certainproteins are measured and compared to the total number of cells or thenumber of protein-expressing cells is determined in a high power fieldunder a light microscope. In one embodiment, the number of integrinbeta7-expressing cells is measured. In one embodiment, the number ofintegrin alphaE-expressing cells is measured. In one embodiment, thenumber of CD3epsilon-expressing cells is measured. In one embodiment,the number of cells expressing a combination of two or three proteinsselected from integrin beta7, integrin alphaE, and CD3epsilon ismeasured. In one embodiment, the biological sample is a tissue biopsysample. In one embodiment, the biopsy is obtained from intestinaltissue. In one embodiment, the biological sample is placed into formalinand optionally embedded in paraffin blocks. In certain embodiments, thenumber of cells expressing one or a combination of integrin beta7,integrin alphaE, or CD3epsilon is measured by an immunohistochemicalmethod. In certain embodiments, the number of cells expressing one or acombination of integrin beta7, integrin alphaE, or CD3epsilon iscompared to the total number of cells in a given area. In oneembodiment, the patient is predicted to respond to therapy comprisingthe integrin beta7 antagonist if the number of cells expressing one ormore of integrin beta7, integrin alphaE, or CD3epsilon is elevated (orhigh) compared to a reference value, for example, a median value. In oneembodiment, the patient is predicted to not respond to therapycomprising the integrin beta7 antagonist if the number of cellsexpressing one or more of integrin beta7, integrin alphaE, or CD3epsilonis low compared to a reference value, for example, a median value. Inone embodiment, the response is clinical remission. In one embodiment,the response is mucosal healing. In one embodiment, the response isclinical response. In certain embodiments, remission in the patient isdetermined to be induced when the absolute Mayo Clinic Score ≤2 and noindividual subscore >1, which is also referred to as clinical remission.In certain embodiments, mucosal healing is determined to have occurredwhen the patient is determined to have an endoscopy subscore of 0 or 1as assessed by flexible sigmoidoscopy. In certain such embodiments,patients who experience mucosal healing are determined to have anendoscopy subscore of 0.

In still another aspect, methods of identifying a patient suffering froma gastrointestinal inflammatory disorder as likely to respond to atherapy comprising an integrin beta7 antagonist are provided. In certainembodiments, the methods comprise: (a) measuring the level of mRNAexpression of at least one gene in a biological sample from the patient,wherein the at least one gene is selected from integrin beta7, integrinalphaE, and CD3epsilon; (b) comparing the level of mRNA expressionmeasured in (a) to a reference level; and (c) identifying the patient asmore likely to respond to the therapy comprising an integrin beta7antagonist when the level of mRNA expression measured in (a) is abovethe reference level. In one embodiment, the patient is a human. In oneembodiment, the patient is a TNF inadequate responder (TNF-IR). In oneembodiment, the gastrointestinal inflammatory disorder is aninflammatory bowel disease. In one embodiment, the inflammatory boweldisease is ulcerative colitis or Crohn's disease. In one embodiment, theinflammatory bowel disease is ulcerative colitis and the response isselected from clinical response, mucosal healing and remission.

In a further aspect, methods of treating a patient having agastrointestinal inflammatory disorder are provided. In certainembodiments, the methods comprise: (a) measuring the level of mRNAexpression of at least one gene in a biological sample from the patient,wherein the at least one gene is selected from integrin beta7, integrinalphaE, and CD3epsilon; (b) comparing the level of mRNA expressionmeasured in (a) to a reference level; (c) identifying the patient asmore likely to respond a therapy comprising an integrin beta7 antagonistwhen the level of mRNA expression measured in (a) is above the referencelevel; and (d) administering the therapy when the level of mRNAexpression measured in (a) is above the reference level, therebytreating the gastrointestinal inflammatory disorder. In one embodiment,100 mg of the integrin beta7 antagonist is administered subcutaneouslyonce every four weeks. In one embodiment, a flat loading dose of 420 mgof the integrin beta7 antagonist is administered subcutaneously,followed two weeks after administration of the loading dose by a secondsubcutaneous administration of 300 mg of the integrin beta7 antagonist,followed two weeks after the second administration by a thirdsubcutaneous administration of 300 mg of the integrin beta7 antagonist,followed four weeks after the third administration by subcutaneousadministration of 300 mg of the integrin beta7 antagonist and thereaftersubcutaneous administration of 300 mg of the integrin beta7 antagonistonce every four weeks. In one embodiment, the patient is a human. In oneembodiment, the patient is a TNF inadequate responder (TNF-IR). In oneembodiment, the gastrointestinal inflammatory disorder is aninflammatory bowel disease. In one embodiment, the inflammatory boweldisease is ulcerative colitis or Crohn's disease. In one embodiment, theinflammatory bowel disease is ulcerative colitis and the response isselected from clinical response, mucosal healing and remission. In oneembodiment, administration of the integrin beta7 antagonist results inone or more of the following: (1) a 3-point decrease and 30% reductionfrom baseline in MCS and ≥1-point decrease in rectal bleeding subscoreor absolute rectal bleeding score of 0 or 1, (2) an endoscopic subscoreof 0 or 1, (3) MCS ≤2 with no individual subscore >1.

In still yet another aspect, methods of predicting the response of apatient suffering from a gastrointestinal inflammatory disorder to atherapy comprising an integrin beta7 antagonist are provided. In certainembodiments, the methods comprise: obtaining a biological sample fromthe patient, measuring the number of cells expressing at least oneprotein selected from integrin beta7, integrin alphaE, and CD3epsilon,comparing the number of expressing cells measured in the sample to areference level, and predicting the response of the patient to thetherapy, wherein elevated numbers of expressing cells compared to thereference level indicates response to the therapy and a low number ofexpressing cells compared to the reference level indicates non-responseto the therapy. In one embodiment, the patient is a human. In oneembodiment, the patient is a TNF inadequate responder (TNF-IR). In oneembodiment, the gastrointestinal inflammatory disorder is aninflammatory bowel disease. In one embodiment, the inflammatory boweldisease is ulcerative colitis or Crohn's disease. In one embodiment, theinflammatory bowel disease is ulcerative colitis and the response isselected from clinical response, mucosal healing and remission. In oneembodiment, the biological sample is intestinal tissue. In oneembodiment, the methods comprise measuring the number of expressingcells by an immunohistochemical method. In one embodiment, the measuringcomprises contacting the sample with an agent that specifically binds tointegrin beta7 protein, integrin alphaE protein, or CD3epsilon proteinand detecting the amount of complex formed, and thereby measuring thenumber of expressing cells. In one embodiment, the number of expressingcells in the sample is divided by the total number of cells in thesample. In one embodiment, the number of expressing cells in a highpower field under a light microscope is determined. In one embodiment,the reference level is a median value.

In another aspect, methods of predicting responsiveness of agastrointestinal inflammatory disorder patient to an integrin beta7antagonist treatment are provided. In certain embodiments, the methodscomprise determining the number of cells expressing at least one proteinselected from integrin beta7, integrin alphaE, and CD3epsilon in abiological sample from the patient, wherein elevated numbers ofexpressing cells compared to a reference level identifies the patient asone who is likely to respond to the integrin beta7 antagonist treatment.In one embodiment, the patient is a human. In one embodiment, thepatient is a TNF inadequate responder (TNF-IR). In one embodiment, thegastrointestinal inflammatory disorder is an inflammatory bowel disease.In one embodiment, the inflammatory bowel disease is ulcerative colitisor Crohn's disease. In one embodiment, the inflammatory bowel disease isulcerative colitis and the response is selected from clinical response,mucosal healing and remission. In one embodiment, the biological sampleis intestinal tissue. In one embodiment, the methods comprise measuringthe number of expressing cells by an immunohistochemical method. In oneembodiment, the measuring comprises contacting the sample with an agentthat specifically binds to integrin beta7 protein, integrin alphaEprotein, or CD3epsilon protein and detecting the amount of complexformed, and thereby measuring the number of expressing cells. In oneembodiment, the number of expressing cells in the sample is divided bythe total number of cells in the sample. In one embodiment, the numberof expressing cells in a high power field under a light microscope isdetermined. In one embodiment, the reference level is a median value.

In yet another aspect, methods of identifying a patient suffering from agastrointestinal inflammatory disorder as likely to respond to a therapycomprising an integrin beta7 antagonist are provided. In certainembodiments, the methods comprise: (a) measuring the number of cellsexpressing at least one protein in a biological sample from the patient,wherein the at least one protein is selected from integrin beta7,integrin alphaE, and CD3epsilon; (b) comparing the number of cellsmeasured in (a) to a reference level; and (c) identifying the patient asmore likely to respond to the therapy comprising an integrin beta7antagonist when the number of cells measured in (a) is above thereference level. In one embodiment, the patient is a human. In oneembodiment, the patient is a TNF inadequate responder (TNF-IR). In oneembodiment, the gastrointestinal inflammatory disorder is aninflammatory bowel disease. In one embodiment, the inflammatory boweldisease is ulcerative colitis or Crohn's disease. In one embodiment, theinflammatory bowel disease is ulcerative colitis and the response isselected from clinical response, mucosal healing and remission. In oneembodiment, the biological sample is intestinal tissue. In oneembodiment, the methods comprise measuring the number of expressingcells by an immunohistochemical method. In one embodiment, the measuringcomprises contacting the sample with an agent that specifically binds tointegrin beta7 protein, integrin alphaE protein, or CD3epsilon proteinand detecting the amount of complex formed, and thereby measuring thenumber of expressing cells. In one embodiment, the number of expressingcells in the sample is divided by the total number of cells in thesample. In one embodiment, the number of expressing cells in a highpower field under a light microscope is determined. In one embodiment,the reference level is a median value.

In yet still another aspect, methods of treating a patient having agastrointestinal inflammatory disorder are provided. In certainembodiments, the method comprise: (a) measuring the number of cellsexpressing at least one protein in a biological sample from the patient,wherein the at least one protein is selected from integrin beta7,integrin alphaE, and CD3epsilon; (b) comparing the number of expressingcells measured in (a) to a reference level; (c) identifying the patientas more likely to respond a therapy comprising an integrin beta7antagonist when the number of expressing cells measured in (a) is abovethe reference level; and (d) administering the therapy when the numberof expressing cells measured in (a) is above the reference level,thereby treating the gastrointestinal inflammatory disorder. In oneembodiment, 100 mg of the integrin beta7 antagonist is administeredsubcutaneously once every four weeks. In one embodiment, a flat loadingdose of 420 mg of the integrin beta7 antagonist is administeredsubcutaneously, followed two weeks after administration of the loadingdose by a second subcutaneous administration of 300 mg of the integrinbeta7 antagonist, followed two weeks after the second administration bya third subcutaneous administration of 300 mg of the integrin beta7antagonist, followed four weeks after the third administration bysubcutaneous administration of 300 mg of the integrin beta7 antagonistand thereafter subcutaneous administration of 300 mg of the integrinbeta7 antagonist once every four weeks. In one embodiment, the patientis a human. In one embodiment, the patient is a TNF inadequate responder(TNF-IR). In one embodiment, the gastrointestinal inflammatory disorderis an inflammatory bowel disease. In one embodiment, the inflammatorybowel disease is ulcerative colitis or Crohn's disease. In oneembodiment, the inflammatory bowel disease is ulcerative colitis and theresponse is selected from clinical response, mucosal healing andremission. In one embodiment, administration of the integrin beta7antagonist results in one or more of the following: (1) a 3-pointdecrease and 30% reduction from baseline in MCS and ≥1-point decrease inrectal bleeding subscore or absolute rectal bleeding score of 0 or 1,(2) an endoscopic subscore of 0 or 1, (3) MCS ≤2 with no individualsubscore >1. In one embodiment, the biological sample is intestinaltissue. In one embodiment, the methods comprise measuring the number ofexpressing cells by an immunohistochemical method. In one embodiment,the measuring comprises contacting the sample with an agent thatspecifically binds to integrin beta7 protein, integrin alphaE protein,or CD3epsilon protein and detecting the amount of complex formed, andthereby measuring the number of expressing cells. In one embodiment, thenumber of expressing cells in the sample is divided by the total numberof cells in the sample. In one embodiment, the number of expressingcells in a high power field under a light microscope is determined. Inone embodiment, the reference level is a median value.

In still yet another aspect, an integrin beta7 antagonist for use intreating a patient having a gastrointestinal inflammatory disorder isprovided. In certain embodiments, the patient is treated or selected fortreatment when the level of mRNA expression of at least one geneselected from integrin beta7, integrin alphaE, and CD3epsilon is above areference level. In certain embodiments, the patient is treated orselected for treatment when the number of cells expressing at least onprotein selected from integrin beta7, integrin alphaE, and CD3epsilon iselevated or high compared to a reference level. In one embodiment, thereference level is a median value. In one embodiment, the integrin beta7antagonist is for use in treating the patient wherein 100 mg isadministered subcutaneously once every four weeks. In one embodiment,the integrin beta7 antagonist is for use in treating the patient whereina flat loading dose of 420 mg of the integrin beta7 antagonist isadministered subcutaneously, followed two weeks after administration ofthe loading dose by a second subcutaneous administration of 300 mg ofthe integrin beta7 antagonist, followed two weeks after the secondadministration by a third subcutaneous administration of 300 mg of theintegrin beta7 antagonist, followed four weeks after the thirdadministration by subcutaneous administration of 300 mg of the integrinbeta7 antagonist and thereafter subcutaneous administration of 300 mg ofthe integrin beta7 antagonist once every four weeks.

In a further aspect, in vitro use of at least one agent thatspecifically binds to a biomarker selected from integrin beta7 mRNA,integrin alphaE mRNA, CD3epsilon mRNA, integrin beta7 protein, integrinalphaE protein, and CD3epsilon protein is provided. In certainembodiments, the at least one agent is used for identifying or selectinga patient having a gastroinflammatory disorder as likely to respond to atherapy comprising an integrin beta7 antagonist, wherein a level ofintegrin beta7, integrin alphaE, and/or CD3epsilon mRNA expression or anumber of integrin beta7-, integrin alphaE-, and/orCD3epsilon-expressing cells above a reference level identifies orselects that the patient is more likely to respond to the therapy. Inone embodiment, the reference level is a median value.

In still another aspect, methods of treating a gastrointestinalinflammatory disorder in a patient are provided. In certain embodiments,a therapeutically effective amount of an integrin beta7 antagonist isadministered to a patient when a biological sample obtained from thepatient has been determined to express elevated mRNA expression levelsof one or more of certain genes. In one embodiment, the sample has beendetermined to express elevated integrin beta7 mRNA compared to themedian value. In one embodiment, the sample has been determined toexpress elevated integrin alphaE mRNA compared to the median value. Inone embodiment, the sample has been determined to express elevatedCD3epsilon mRNA compared to the median value. In one embodiment, thesample has been determined to express elevated mRNA levels of two orthree of integrin beta7, integrin alphaE, and CD3epsilon compared to themedian levels of the same mRNAs. In certain embodiments, the patient hasbeen selected for treatment based on elevated mRNA expression levels ofcertain genes in the biological sample compared to a median value of thesame gene or genes. In one embodiment, the patient has been selected fortreatment based on elevated integrin beta7 mRNA expression compared tothe median value. In one embodiment, the patient has been selected fortreatment based on elevated integrin alphaE mRNA expression compared tothe median value. In one embodiment, the patient has been selected fortreatment based on elevated CD3epsilon mRNA expression compared to themedian value. In one embodiment, the patient has been selected fortreatment based on elevated mRNA expression of two or three of integrinbeta7, integrin alphaE, and CD3epsilon compared to the median values forthe same mRNAs. In one embodiment, the biological sample is a tissuebiopsy sample. In one embodiment, the biological sample is peripheralwhole blood. In one embodiment, the peripheral whole blood is collectedin a PAXgene tube. In certain embodiments, the mRNA expression level ismeasured by a PCR method. In one embodiment, the PCR method is qPCR. Incertain embodiments administration of the integrin beta7 antagonistresults in one or more of the following: (1) a 3-point decrease and 30%reduction from baseline in MCS and ≥1-point decrease in rectal bleedingsubscore or absolute rectal bleeding score of 0 or 1, (2) an endoscopicsubscore of 0 or 1, (3) MCS ≤2 with no individual subscore >1. In oneembodiment, 100 mg of the integrin beta7 antagonist is administeredsubcutaneously once every four weeks. In one embodiment, a flat loadingdose of 420 mg of the integrin beta7 antagonist is administeredsubcutaneously, followed two weeks after administration of the loadingdose by a second subcutaneous administration of 300 mg of the integrinbeta7 antagonist, followed two weeks after the second administration bya third subcutaneous administration of 300 mg of the integrin beta7antagonist, followed four weeks after the third administration bysubcutaneous administration of 300 mg of the integrin beta7 antagonistand thereafter subcutaneous administration of 300 mg of the integrinbeta7 antagonist once every four weeks.

In certain of the above embodiments, the gastrointestinal inflammatorydisorder is an inflammatory bowel disease, and in certain suchembodiments, the inflammatory bowel disease is ulcerative colitis (UC)or Crohn's disease (CD), and in certain such embodiments, the integrinbeta7 antagonist is a monoclonal anti-beta7 antibody. In certain suchembodiments, the anti-beta7 antibody is selected from a chimericantibody, a human antibody, and a humanized antibody. In certainembodiments, the anti-beta7 antibody is an antibody fragment. In certainembodiments, the anti-beta7 antibody comprises six hypervariable regions(HVRs), wherein:

(i) HVR-L1 comprises amino acid sequence A1-A11, wherein A1-A11 isRASESVDTYLH (SEQ ID NO:1); RASESVDSLLH (SEQ ID NO:7), RASESVDTLLH (SEQID NO:8), or RASESVDDLLH (SEQ ID NO:9) or a variant of SEQ ID NOs:1, 7,8 or 9 (SEQ ID NO:26) wherein amino acid A2 is selected from the groupconsisting of A, G, S, T, and V and/or amino acid A3 is selected fromthe group consisting of S, G, I, K, N, P, Q, R, and T, and/or A4 isselected from the group consisting of E, V, Q, A, D, G, H, I, K, L, N,and R, and/or amino acid A5 is selected from the group consisting of S,Y, A, D, G, H, I, K, N, P, R, T, and V, and/or amino acid A6 is selectedfrom the group consisting of V, R, I, A, G, K, L, M, and Q, and/or aminoacid A7 is selected from the group consisting of D, V, S, A, E, G, H, I,K, L, N, P, S, and T, and/or amino acid A8 is selected from the groupconsisting of D, G, N, E, T, P and S, and/or amino acid A9 is selectedfrom the group consisting of L, Y, I and M, and/or amino acid A10 isselected from the group consisting of L, A, I, M, and V and/or aminoacid A11 is selected from the group consisting of H, Y, F, and S;

(ii) HVR-L2 comprises amino acid sequence B1-B8, wherein B1-B8 isKYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID NO:20), or XaaYASQSIS (SEQ IDNO:21, where Xaa represents any amino acid) or a variant of SEQ IDNOs:2, 20 or 21 (SEQ ID NO:27) wherein amino acid B1 is selected fromthe group consisting of K, R, N, V, A, F, Q, H, P, I, L, Y and Xaa(where Xaa represents any amino acid), and/or amino acid B4 is selectedfrom the group consisting of S and D, and/or amino acid B5 is selectedfrom the group consisting of Q and S, and/or amino acid B6 is selectedfrom the group consisting of S, D, L, and R, and/or amino acid B7 isselected from the group consisting of I, V, E, and K;

(iii) HVR-L3 comprises amino acid sequence C1-C9, wherein C1-C9 isQQGNSLPNT (SEQ ID NO:3) or a variant of SEQ ID NO:3 (SEQ ID NO:28)wherein amino acid C8 is selected from the group consisting of N, V, W,Y, R, S, T, A, F, H, I L, and M;

(iv) HVR-H1 comprises amino acid sequence D1-D10 wherein D1-D10 isGFFITNNYWG (SEQ ID NO:4);

(v) HVR-H2 comprises amino acid sequence E1-E17 wherein E1-E17 isGYISYSGSTSYNPSLKS (SEQ ID NO:5), or a variant of SEQ ID NO:5 (SEQ IDNO:29) wherein amino acid E2 is selected from the group consisting of Y,F, V, and D, and/or amino acid E6 is selected from the group consistingof S and G, and/or amino acid E10 is selected from the group consistingof S and Y, and/or amino acid E12 is selected from the group consistingof N, T, A, and D, and/or amino acid 13 is selected from the groupconsisting of P, H, D, and A, and/or amino acid E15 is selected from thegroup consisting of L and V, and/or amino acid E17 is selected from thegroup consisting of S and G; and

(vi) HVR-H3 comprises amino acid sequence F2-F11 wherein F2-F11 isMTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF (SEQ ID NO:19); or comprisesamino acid sequence F1-F11, wherein F1-F11 is AMTGSSGYFDF (SEQ IDNO:16), ARTGSSGYFDF (SEQ ID NO:17), or AQTGSSGYFDF (SEQ ID NO:18), or avariant of SEQ ID NOs:6, 16, 17, 18, or 19 (SEQ ID NO:30) wherein aminoacid F2 is R, M, A, E, G, Q, S, and/or amino acid F11 is selected fromthe group consisting of F and Y. In certain such embodiments, theanti-beta7 antibody comprises three heavy chain hypervariable region(HVR-H1-H3) sequences and three light chain hypervariable region(HVR-L1-L3) sequences, wherein:

-   -   (i) HVR-L1 comprises SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9;    -   (ii) HVR-L2 comprises SEQ ID NO:2;    -   (iii) HVR-L3 comprises SEQ ID NO:3;    -   (iv) HVR-H1 comprises SEQ ID NO:4;    -   (v) HVR-H2 comprises SEQ ID NO:5; and    -   (vi) HVR-H3 comprises SEQ ID NO:6 or SEQ ID NO:16 or SEQ TD        NO:17 or SEQ ID NO:19.        In certain embodiments, the anti-beta7 antibody comprises a        variable light chain comprising the amino acid sequence of SEQ        ID NO:24. In certain embodiments, the anti-beta7 antibody        comprises a variable heavy chain comprising the amino acid        sequence of SEQ ID NO:31 (SEQ ID NO:31 corresponds to SEQ ID        NO:25 except that the amino acid sequence of HVR-H3 of SEQ ID        NO:25 (MTGSSGYFDF (SEQ ID NO:6) or AMTGSSGYFDF (SEQ ID NO:16))        is substituted with RTGSSGYFDF (SEQ ID NO:19) or ARTGSSGYFDF        (SEQ ID NO:17), respectively). In certain embodiments, the        anti-beta7 antibody comprises a variable light chain comprising        the amino acid sequence of SEQ ID NO:24 and a variable heavy        chain comprising the amino acid sequence of SEQ ID NO:31. In        certain embodiments, the anti-beta7 antibody is rhuMAb Beta7,        also referred to herein as etrolizumab.

In another aspect, the integrin beta7 antagonist is a monoclonalanti-alpha4/beta7 antibody. In certain embodiments, theanti-alpha4/beta7 antibody is vedolizumab. In certain embodiments, theanti-alpha4/beta7 antibody is AMG 181.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows alignment of sequences of the variable light andheavy chains for the following consensus sequences and anti-beta7subunit antibody sequences: light chain human subgroup kappa I consensussequence (FIG. 1A, SEQ ID NO:12), heavy chain human subgroup IIIconsensus sequence (FIG. 1B, SEQ ID NO: 13), rat anti-mouse beta7antibody (Fib504) variable light chain (FIG. 1A, SEQ ID NO:10), ratanti-mouse beta7 antibody (Fib504) variable heavy chain (FIG. 1B, SEQ IDNO: 11), and humanized antibody variants: Humanized hu504Kgraft variablelight chain (FIG. 1A, SEQ ID NO: 14), humanized hu504K graft variableheavy chain (FIG. 1B, SEQ ID NO: 15), variants hu504-5, hu504-16, andhu504-32 (amino acid variations from humanized hu504K graft areindicated in FIG. 1A) (light chain) (SEQ ID NOS:22-24, respectively, inorder of appearance) and FIG. 1B (heavy chain) for variants hu504-5,hu504-16, and hu504-32 (SEQ ID NO:25).

FIG. 2 shows the study schema for the Phase II clinical study asdescribed in Example 1.

FIGS. 3A-3C show the percent of patients treated with placebo (stippledbars), 100 mg/dose etrolizumab (hatched bars), or 300 mg/doseetrolizumab (open bars) in remission at week 10 (FIG. 3A), showingmucosal healing at week 10 (FIG. 3B), or showing clinical response atweek 10 (FIG. 3C), stratified by baseline beta7 gene expression levels(low, below the median vs. high, above the median) in intestinalbiopsies as described in Example 2.

FIGS. 4A-4C show the percent of patients treated with placebo (stippledbars), 100 mg/dose etrolizumab (hatched bars), or 300 mg/doseetrolizumab (open bars) in remission at week 10 (FIG. 4A), showingmucosal healing at week 10 (FIG. 4B), or showing clinical response atweek 10 (FIG. 4C), stratified by baseline CD3epsilon gene expressionlevels (low, below the median vs. high, above the median) in intestinalbiopsies as described in Example 2.

FIG. 5 shows the correlation of beta7 expression levels as detected byFACS analysis in CD45+ cells, CD3+ cells and CD19+ cells to the level ofbeta7 gene expression in peripheral blood as detected by qPCR in bothIBD patients (open dots) and in healthy controls (filled dots) asdescribed in Example 2.

FIGS. 6A-6C show the percent of patients treated with placebo (stippledbars), 100 mg/dose etrolizumab (hatched bars), or 300 mg/doseetrolizumab (open bars) in remission at week 10 (FIG. 6A), showingmucosal healing at week 10 (FIG. 6B), or showing clinical response atweek 10 (FIG. 6C), stratified by baseline beta7 gene expression levels(low, below the median vs. high, above the median) in peripheral bloodas described in Example 2.

FIGS. 7A-7G show integrin alphaE expression in intestinal biopsiesdetermined by immunohistochemistry or by qPCR as described in Example 2.(FIG. 7A) AlphaE immunohistochemistry in colon (left panels) or ileum(right panels) tissue obtained from non-IBD patients (top panels),Crohn's disease patients (middle panels), or a UC patient (bottom panel;in each case, positively-staining regions are darkest; (FIG. 7B)computerized autocounts of alphaE+ cells as a percentage of total cellsin colon, ileum, or jejunum tissue as indicated, from non-IBD patients,UC patients, and CD patients; (FIG. 7C) alphaE gene expression relativeto GAPDH in full thickness biopsies from the colon non-IBD, UC and thecolon, ileum or jejunum of Crohn's disease patients as indicatedundergoing intestinal resections; (FIG. 7D) computerized autocounts ofalphaE+ cells as a percentage of total cells in biopsy tissue obtainedfrom patients enrolled in the Phase II etrolizumab trial at screeningand identified as TNF naïve (left side of graph) or TNF-IR (right sideof graph), stippled dots: etrolizumab remitters, open dots: etrolizumabnon-remitters, black dots: placebo, dotted line indicates median; (FIG.7E) exemplary alphaE staining in screening biopsies from patientsenrolled in the the Phase II etrolizumab trial, left panel: alphaE highpatient, right panel: alphaE low patient; in each case,positively-staining regions are darkest; (FIG. 7F) alphaE geneexpression relative to GAPDH expression in biopsy tissue obtained frompatients enrolled in the Phase II etrolizumab trial at screening andidentified as TNF naïve (left side of graph) or TNF-IR (right side ofgraph), stippled dots: etrolizumab remitters, open dots: etrolizumabnon-remitters, black dots: placebo, dotted line indicates median; (FIG.7G) graph showing the relationship of alphaE gene expression as measuredby qPCR (vertical axis) to alphaE protein expression as measured by IHC(horizontal axis); stippled dots: etrolizumab remitters, open dots:etrolizumab non-remitters, dotted line indicates median.

FIGS. 8A-8F show the proportion of patients (percentage) stratified bybaseline alphaE gene expression levels (low, below the median vs. high,above the median) in intestinal biopsies and treated with placebo(stippled bars), 100 mg/dose etrolizumab (hatched bars), or 300 mg/doseetrolizumab (open bars) that were in remission at week 10 (FIG. 8A),showing mucosal healing at week 10 (FIG. 8B), or showing clinicalresponse at week 10 (FIG. 8C); or stratified by baseline alphaE proteinexpression (low, below the median vs. high, above the median) inintestinal biopsies and treated with placebo (stippled bars), 100mg/dose etrolizumab (hatched bars), or 300 mg/dose etrolizumab (openbars) that were in remission at week 10 (FIG. 8D), showing mucosalhealing at week 10 (FIG. 8E), or showing clinical response at week 10(FIG. 8F), as described in Example 2.

FIGS. 9A-9F shows the proportion of TNF-naïve patients (percentage)stratified by baseline alphaE gene expression levels (low, below themedian vs. high, above the median) in intestinal biopsies and treatedwith placebo (stippled bars), 100 mg/dose etrolizumab (hatched bars), or300 mg/dose etrolizumab (open bars) that were in remission at week 10(FIG. 9A), showing mucosal healing at week 10 (FIG. 9B), or showingclinical response at week 10 (FIG. 9C); or stratified by baseline alphaEprotein expression (low, below the median vs. high, above the median) inintestinal biopsies and treated with placebo (stippled bars), 100mg/dose etrolizumab (hatched bars), or 300 mg/dose etrolizumab (openbars) that were in remission at week 10 (FIG. 9D), showing mucosalhealing at week 10 (FIG. 9E), or showing clinical response at week 10(FIG. 9F), as described in Example 2.

FIGS. 10A-10F show the proportion of patients (percentage) stratified bybaseline alphaE gene expression levels (low, below the median vs. high,above the median) in peripheral blood at screening and treated withplacebo (stippled bars), 100 mg/dose etrolizumab (hatched bars), or 300mg/dose etrolizumab (open bars) that were in remission at week 10 (FIG.10A), showing mucosal healing at week 10 (FIG. 10B), or showing clinicalresponse at week 10 (FIG. 10C); or stratified by alphaE gene expression(low, below the median vs. high, above the median) in peripheral bloodat day 1 and treated with placebo (stippled bars), 100 mg/doseetrolizumab (hatched bars), or 300 mg/dose etrolizumab (open bars) thatwere in remission at week 10 (FIG. 10D), showing mucosal healing at week10 (FIG. 10E), or showing clinical response at week 10 (FIG. 10F), asdescribed in Example 2.

FIGS. 11A-11F show the proportion of TNF naïve patients (percentage)stratified by baseline alphaE gene expression levels (low, below themedian vs. high, above the median) in peripheral blood at screening andtreated with placebo (stippled bars), 100 mg/dose etrolizumab (hatchedbars), or 300 mg/dose etrolizumab (open bars) that were in remission atweek 10 (FIG. 11A), showing mucosal healing at week 10 (FIG. 11B), orshowing clinical response at week 10 (FIG. 11C); or stratified by alphaEgene expression (low, below the median vs. high, above the median) inperipheral blood at day 1 and treated with placebo (stippled bars), 100mg/dose etrolizumab (hatched bars), or 300 mg/dose etrolizumab (openbars) that were in remission at week 10 (FIG. 11D), showing mucosalhealing at week 10 (FIG. 11E), or showing clinical response at week 10(FIG. 11F), as described in Example 2.

FIG. 12 shows the study schema for the Phase II open label extensionclinical study as described in Example 1.

FIGS. 13A-13F show the percentage of TNF-IR patients from the open labelextension study stratified by baseline alphaE gene expression (low,below the median vs. high, above the median) in intestinal biopsies thatwere in clinical remission (FIG. 13A) or showing clinical response (FIG.13B); or stratified by baseline alphaE protein expression (low, belowthe median vs. high, above the median) in intestinal biopsies that werein clinical remission (FIG. 13C) or showing clinical response (FIG.13D); or stratified by alphaE gene expression in peripheral blood thatwere in clinical remission (FIG. 13E) or showing clinical response (FIG.13F).

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provideone skilled in the art with a general guide to many of the terms used inthe present application.

Certain Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with any document incorporatedherein by reference, the definition set forth below shall control.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a protein”includes a plurality of proteins; reference to “a cell” includesmixtures of cells, and the like.

Ranges provided in the specification and appended claims include bothend points and all points between the end points. Thus, for example, arange of 2.0 to 3.0 includes 2.0, 3.0, and all points between 2.0 and3.0.

“Treatment,” “treating,” and grammatical variations thereof refer toclinical intervention in an attempt to alter the natural course of theindividual or cell being treated, and can be performed either forprophylaxis or during the course of clinical pathology. Desirableeffects of treatment include preventing occurrence or recurrence ofdisease, alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

“Treatment regimen” refers to a combination of dosage, frequency ofadministration, or duration of treatment, with or without addition of asecond medication.

“Effective treatment regimen” refers to a treatment regimen that willoffer beneficial response to a patient receiving the treatment.

“Patient response” or “patient responsiveness” can be assessed using anyendpoint indicating a benefit to the patient, including, withoutlimitation, (1) inhibition, to some extent, of disease progression,including slowing down and complete arrest; (2) reduction in the numberof disease episodes and/or symptoms; (3) reduction in lesional size; (4)inhibition (i.e., reduction, slowing down or complete stopping) ofdisease cell infiltration into adjacent peripheral organs and/ortissues; (5) inhibition (i.e., reduction, slowing down or completestopping) of disease spread; (6) decrease of auto-immune response, whichmay, but does not have to, result in the regression or ablation of thedisease lesion; (7) relief, to some extent, of one or more symptomsassociated with the disorder; (8) increase in the length of disease-freepresentation following treatment; and/or (9) decreased mortality at agiven point of time following treatment. The term “responsiveness”refers to a measurable response, including complete response (CR) andpartial response (PR).

As used herein, “complete response” or “CR” means the disappearance ofall signs of inflammation or remission in response to treatment. Thisdoes not necessarily mean the disease has been cured.

“Partial response” or “PR” refers to a decrease of at least 50% in theseverity of inflammation, in response to treatment.

A “beneficial response” of a patient to treatment with an integrin beta7antagonist and similar wording refers to the clinical or therapeuticbenefit imparted to a patient at risk for or suffering from agastrointestinal inflammatory disorder from or as a result of thetreatment with the antagonist, such as an anti-beta7 integrin antibody.Such benefit includes cellular or biological responses, a completeresponse, a partial response, a stable disease (without progression orrelapse), or a response with a later relapse of the patient from or as aresult of the treatment with the antagonist.

As used herein, “non-response” or “lack of response” or similar wordingmeans an absence of a complete response, a partial response, or abeneficial response to treatment with an integrin beta7 antagonist.

“A patient maintains responsiveness to a treatment” when the patient'responsiveness does not decrease with time during the course of atreatment.

The term “sample,” or “test sample” as used herein, refers to acomposition that is obtained or derived from a subject of interest thatcontains a cellular and/or other molecular entity that is to becharacterized and/or identified, for example based on physical,biochemical, chemical and/or physiological characteristics. In oneembodiment, the definition encompasses blood and other liquid samples ofbiological origin and tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom. The source of the tissue sample maybe solid tissue as from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids; and cells from any time in gestation or development of thesubject or plasma. The term “sample,” or “test sample” includesbiological samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or polynucleotides,or embedding in a semi-solid or solid matrix for sectioning purposes.For the purposes herein a “section” of a tissue sample is meant a singlepart or piece of a tissue sample, e.g. a thin slice of tissue or cellscut from a tissue sample. Samples include, but are not limited to, wholeblood, blood-derived cells, serum, plasma, lymph fluid, synovial fluid,cellular extracts, and combinations thereof. In one embodiment, thesample is a clinical sample. In another embodiment, the sample is usedin a diagnostic assay.

A “reference sample,” as used herein, refers to any sample, standard, orlevel that is used for comparison purposes. In one embodiment, areference sample is obtained from a healthy and/or non-diseased part ofthe body (e.g., tissue or cells) of the same subject or patient. Inanother embodiment, a reference sample is obtained from an untreatedtissue and/or cell of the body of the same subject or patient. In yetanother embodiment, a reference sample is obtained from a healthy and/ornon-diseased part of the body (e.g., tissues or cells) of an individualwho is not the subject or patient. In even another embodiment, areference sample is obtained from an untreated tissue and/or cell partof the body of an individual who is not the subject or patient.

“A beta7 integrin antagonist” or “beta7 antagonist” refers to anymolecule that inhibits one or more biological activities or blockingbinding of beta7 integrin with one or more of its associated molecules.Antagonists of the invention can be used to modulate one or more aspectsof beta7 associated effects, including but not limited to associationwith alpha4 integrin subunit, association with alphaE integrin subunit,binding of alpha4beta7 integrin to MAdCAM, VCAM-1 or fibronectin andbinding of alphaEbeta7 integrin to E-cadherin. These effects can bemodulated by any biologically relevant mechanism, including disruptionof ligand binding to beta7 subunit or to the alpha4beta7 or alphaEbeta7dimeric integrin, and/or by disrupting association between the alpha andbeta integrin subunits such that formation of the dimeric integrin isinhibited. In one embodiment of the invention, the beta7 antagonist isan anti-beta7 integrin antibody (or anti-beta7 antibody). In oneembodiment, the anti-beta7 integrin antibody is a humanized anti-beta7integrin antibody and more particularly a recombinant humanizedmonoclonal anti-beta7 antibody (or rhuMAb beta7). In some embodiments,the anti-beta7 antibodies of the present invention are anti-integrinbeta7 antagonistic antibodies that inhibit or block the binding of beta7subunit with alpha4 integrin subunit, association with alphaE integrinsubunit, binding of alpha4beta7 integrin to MAdCAM, VCAM-1 orfibronectin and binding of alphaEbeta7 integrin to E-cadherin.

By “beta7 subunit” or “β7 subunit” is meant the human β37 integrinsubunit (Erle et al., (1991) J. Biol. Chem. 266:11009-11016). The beta7subunit associates with alpha4 integrin subunit, such as the human.alpha.4 subunit (Kilger and Holzmann (1995) J. Mol. Biol. 73:347-354).The alpha4beta7 integrin is reportedly expressed on a majority of maturelymphocytes, as well as a small population of thymocytes, bone marrowcells and mast cells. (Kilshaw and Murant (1991) Eur. J. Immunol.21:2591-2597; Gurish et al., (1992) 149: 1964-1972; and Shaw, S. K. andBrenner, M. B. (1995) Semin. Immunol. 7:335). The beta7 subunit alsoassociates with the alphaE subunit, such as the human alphaE integrinsubunit (Cepek, K. L, et al. (1993) J. Immunol. 150:3459). ThealphaEbeta7 integrin is expressed on intra-intestinal epitheliallymphocytes (iIELs) (Cepek, K. L. (1993) supra).

By “alphaE subunit” or “alphaE integrin subunit” or “αE subunit” or “αEintegrin subunit” or “CD103” is meant an integrin subunit found to beassociated with beta7 integrin on intra-epithelial lymphocytes, whichalphaEbeta7 integrin mediates binding of the iELs to intestinalepithelium expressing E-cadherin (Cepek, K. L. et al. (1993) J. Immunol.150:3459; Shaw, S. K. and Brenner, M. B. (1995) Semin. Immunol. 7:335).

“MAdCAM” or “MAdCAM-1” are used interchangeably in the context of thepresent invention and refer to the protein mucosal addressin celladhesion molecule-1, which is a single chain polypeptide comprising ashort cytoplasmic tail, a transmembrane region and an extracellularsequence composed of three immunoglobulin-like domains. The cDNAs formurine, human and macaque MAdCAM-1 have been cloned (Briskin, et al.,(1993) Nature, 363:461-464; Shyjan et al., (1996) J. Immunol.156:2851-2857).

“VCAM-1” or “vascular cell adhesion molecule-1” “CD106” refers to aligand of alpha4beta7 and alpha4beta1, expressed on activatedendothelium and important in endothelial-leukocyte interactions such asbinding and transmigration of leukocytes during inflammation.

“CD45” refers to a protein of the protein tyrosine phosphatase (PTP)family. PTPs are known to be signaling molecules that regulate a varietyof cellular processes including cell growth, differentiation, mitoticcycle, and oncogenic transformation. This PTP contains an extracellulardomain, a single transmembrane segment and two tandem intracytoplasmiccatalytic domains, and thus belongs to receptor type PTP. This gene isspecifically expressed in hematopoietic cells. This PTP has been shownto be an essential regulator of T- and B-cell antigen receptorsignaling. It functions through either direct interaction withcomponents of the antigen receptor complexes, or by activating variousSrc family kinases required for the antigen receptor signaling. This PTPalso suppresses JAK kinases, and thus functions as a regulator ofcytokine receptor signaling. Four alternatively spliced transcriptsvariants of this gene, which encode distinct isoforms, have beenreported. (Tchilian E Z, Beverley P C (2002). “CD45 in memory anddisease.” Arch. Immunol. Ther. Exp. (Warsz.) 50 (2): 85-93. Ishikawa H,Tsuyama N, Abroun S, et al. (2004). “Interleukin-6, CD45 and thesrc-kinases in myeloma cell proliferation.” Leuk. Lymphoma 44(9):1477-81.

Various isoforms of CD45 exist: CD45RA, CD45RB, CD45RC, CD45RAB,CD45RAC, CD45RBC, CD45RO, CD45R (ABC). CD45 is also highly glycosylated.CD45R is the longest protein and migrates at 200 kDa when isolated fromT cells. B cells also express CD45R with heavier glycosylation, bringingthe molecular weight to 220 kDa, hence the name B220; B cell isoform of220 kDa. B220 expression is not restricted to B cells and can also beexpressed on activated T cells, on a subset of dendritic cells and otherantigen presenting cells. Stanton T, Boxall S, Bennett A, et al. (2004).“CD45 variant alleles: possibly increased frequency of a novel exon 4CD45 polymorphism in HIV seropositive Ugandans.” Immunogenetics 56 (2):107-10.

“Gut-homing lymphocytes” refer to a subgroup of lymphocytes having thecharacteristic of selectively homing to intestinal lymph nodes andtissues but not homing to peripheral lymph nodes and tissues. Thissubgroup of lymphocytes are characterized by an unique expressionpattern of a combination of multiples cell surface molecules, including,but not limited to, the combination of CD4, CD45RA and Beta7. Typically,at least two subsets of peripheral blood CD4⁺ lymphocytes can besubdivided based on the markers of CD45RA and Beta7,CD45RA^(−β)7^(high), and CD45RA⁻β7^(low) CD4⁺ cells. CD45RA⁻β7^(high)CD4⁺ cells home preferentially to intestinal lymph nodes and tissues,whereas CD45RA^(−β)7^(low) CD4⁺ cells home preferentially to peripherallymph nodes and tissues (Rott et al. 1996; Rott et al. 1997; Williams etal. 1998; Rosé et al. 1998; Williams and Butcher 1997; Butcher et al.1999). Gut-homing lymphocytes are therefore a distinctive subgroup oflymphocytes identified as CD45RA^(−β)7^(high) CD4⁺ in a flow cytometryassay. The methods of identifying this group of lymphocytes arewell-known in the art.

As used herein with respect to a cell surface marker, the symbol “+”indicates a positive expression of a cell surface marker. For instance,CD4⁺ lymphocytes are a group of lymphocytes having CD4 expressed ontheir cell surfaces.

As used herein with respect to a cell surface marker, the symbol “−”indicates a negative expression of a cell surface marker. For instance,CD45RA⁻ lymphocytes are a group of lymphocytes having no CD45RAexpressed on their cell surfaces.

“Gastrointestinal inflammatory disorders” are a group of chronicdisorders that cause inflammation and/or ulceration in the mucousmembrane. These disorders include, for example, inflammatory boweldisease (e.g., Crohn's disease, ulcerative colitis, indeterminatecolitis and infectious colitis), mucositis (e.g., oral mucositis,gastrointestinal mucositis, nasal mucositis and proctitis), necrotizingenterocolitis and esophagitis.

“Inflammatory Bowel Disease” or “IBD” is used interchangeably herein torefer to diseases of the bowel that cause inflammation and/or ulcerationand includes without limitation Crohn's disease and ulcerative colitis.

“Crohn's disease (CD)” and “ulcerative colitis (UC)” are chronicinflammatory bowel diseases of unknown etiology. Crohn's disease, unlikeulcerative colitis, can affect any part of the bowel. The most prominentfeature Crohn's disease is the granular, reddish-purple edematousthickening of the bowel wall. With the development of inflammation,these granulomas often lose their circumscribed borders and integratewith the surrounding tissue. Diarrhea and obstruction of the bowel arethe predominant clinical features. As with ulcerative colitis, thecourse of Crohn's disease may be continuous or relapsing, mild orsevere, but unlike ulcerative colitis, Crohn's disease is not curable byresection of the involved segment of bowel. Most patients with Crohn'sdisease require surgery at some point, but subsequent relapse is commonand continuous medical treatment is usual.

Crohn's disease may involve any part of the alimentary tract from themouth to the anus, although typically it appears in the ileocolic,small-intestinal or colonic-anorectal regions. Histopathologically, thedisease manifests by discontinuous granulomatomas, crypt abscesses,fissures and aphthous ulcers. The inflammatory infiltrate is mixed,consisting of lymphocytes (both T and B cells), plasma cells,macrophages, and neutrophils. There is a disproportionate increase inIgM- and IgG-secreting plasma cells, macrophages and neutrophils.

Anti-inflammatory drugs sulfasalazine and 5-aminosalisylic acid (5-ASA)are used for treating mildly active colonic Crohn's disease and arecommonly prescribed in an attempt to maintain remission of the disease.Metroidazole and ciprofloxacin are similar in efficacy to sulfasalazineand are particularly prescribed for treating perianal disease. In moresevere cases, corticosteroids are prescribed to treat activeexacerbations and can sometimes maintain remission. Azathioprine and6-mercaptopurine have also been used in patients who require chronicadministration of corticosteroids. It has been suggested that thesedrugs may play a role in the long-term prophylaxis. Unfortunately, therecan be a very long delay (up to six months) before onset of action insome patients. Antidiarrheal drugs can also provide symptomatic reliefin some patients. Nutritional therapy or elemental diet can improve thenutritional status of patients and induce symptomatic improvement ofacute disease, but it does not induce sustained clinical remissions.Antibiotics are used in treating secondary small bowel bacterialovergrowth and in treatment of pyogenic complications.

“Ulcerative colitis (UC)” afflicts the large intestine. The course ofthe disease may be continuous or relapsing, mild or severe. The earliestlesion is an inflammatory infiltration with abscess formation at thebase of the crypts of Lieberkuhn. Coalescence of these distended andruptured crypts tends to separate the overlying mucosa from its bloodsupply, leading to ulceration. Symptoms of the disease include cramping,lower abdominal pain, rectal bleeding, and frequent, loose dischargesconsisting mainly of blood, pus and mucus with scanty fecal particles. Atotal colectomy may be required for acute, severe or chronic,unremitting ulcerative colitis.

The clinical features of UC are highly variable, and the onset may beinsidious or abrupt, and may include diarrhea, tenesmus and relapsingrectal bleeding. With fulminant involvement of the entire colon, toxicmegacolon, a life-threatening emergency, may occur. Extraintestinalmanifestations include arthritis, pyoderma gangrenoum, uveitis, anderythema nodosum.

Treatment for UC includes sulfasalazine and relatedsalicylate-containing drugs for mild cases and corticosteroid drugs insevere cases. Topical administration of either salicylates orcorticosteroids is sometimes effective, particularly when the disease islimited to the distal bowel, and is associated with decreased sideeffects compared with systemic use. Supportive measures such asadministration of iron and antidiarrheal agents are sometimes indicated.Azathioprine, 6-mercaptopurine and methotrexate are sometimes alsoprescribed for use in refractory corticosteroid-dependent cases.

An “effective dosage” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

As used herein, the term “patient” refers to any single subject forwhich treatment is desired. In certain embodiments, the patient hereinis a human.

A “subject” herein is typically a human. In certain embodiments, asubject is a non-human mammal. Exemplary non-human mammals includelaboratory, domestic, pet, sport, and stock animals, e.g., mice, cats,dogs, horses, and cows. Typically, the subject is eligible fortreatment, e.g., treatment of a gastrointestinal inflammatory disorder.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for example, fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, and typically mostor all, of the functions normally associated with that portion whenpresent in an intact antibody. In one embodiment, an antibody fragmentcomprises an antigen binding site of the intact antibody and thusretains the ability to bind antigen. In another embodiment, an antibodyfragment, for example one that comprises the Fc region, retains at leastone of the biological functions normally associated with the Fc regionwhen present in an intact antibody, such as FcRn binding, antibody halflife modulation, ADCC function and complement binding. In oneembodiment, an antibody fragment is a monovalent antibody that has an invivo half life substantially similar to an intact antibody. For example,such an antibody fragment may comprise on antigen binding arm linked toan Fc sequence capable of conferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin lo sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which comprises an amino acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. Such techniques include screening human-derivedcombinatorial libraries, such as phage display libraries (see, e.g.,Marks et al., J. Mol. Biol., 222: 581-597 (1991) and Hoogenboom et al.,Nucl. Acids Res., 19: 4133-4137 (1991)); using human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies (see, e.g., Kozbor J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 55-93 (Marcel Dekker, Inc., New York, 1987); andBoerner et al., J. Immunol., 147: 86 (1991)); and generating monoclonalantibodies in transgenic animals (e.g., mice) that are capable ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature,362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993)).This definition of a human antibody specifically excludes a humanizedantibody comprising antigen-binding residues from a non-human animal.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and often more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or nonreducing conditions usingCoomassie blue or silver stain. Isolated antibody includes the antibodyin situ within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of theseHVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 49-56 or 50-56 or 52-56 (L2) and89-97 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102,94-102 or 95-102 (H3) in the VH. The variable domain residues arenumbered according to Kabat et al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In certain embodiments, affinitymatured antibodies will have nanomolar or even picomolar affinities forthe target antigen. Affinity matured antibodies are produced byprocedures known in the art. Marks et al. Bio/Technology 10:779-783(1992) describes affinity maturation by VH and VL domain shuffling.Random mutagenesis of CDR and/or framework residues is described by:Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier etal. Gene 169:147-155 (1996); Yelton et al. J. Immunol. 155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins etal. J. Mol. Biol. 226:889-896 (1992).

The phrase “substantially similar,” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention.

The term “variable” in connection with antibodies or immunoglobulinsrefers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not evenly distributed throughout thevariable domains of antibodies. It is concentrated in three segmentscalled hypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FRs). The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the j-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab=fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (W. B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

Unless indicated otherwise, herein the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991), expresslyincorporated herein by reference. The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

A “functional Fc region” possesses an “effector function” of a nativesequence Fe region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g., an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. In certain embodiments, the variant Fc region has atleast one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g., from about oneto about ten amino acid substitutions, and in certain embodiments fromabout one to about five amino acid substitutions in a native sequence Fcregion or in the Fc region of the parent polypeptide. In certainembodiments, the variant Fc region herein will possess at least about80% homology with a native sequence Fc region and/or with an Fc regionof a parent polypeptide, or at least about 90% homology therewith, or atleast about 95% homology therewith.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils. The effector cells may be isolated from a native sourcethereof, e.g., from blood or PBMCs as described herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In certain embodiments, FcR is anative sequence human FcR. Moreover, FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITM) in its cytoplasmic domain (seereview M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and regulateshomeostasis of immunoglobulins. Antibodies with improved binding to theneonatal Fc receptor (FcRn), and increased half-lives, are described inWO00/42072 (Presta, L.) and US2005/0014934A1 (Hinton et al.). Theseantibodies comprise an Fc region with one or more substitutions thereinwhich improve binding of the Fc region to FcRn. For example, the Fcregion may have substitutions at one or more of positions 238, 250, 256,265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362,376, 378, 380, 382, 413, 424, 428 or 434 (Eu numbering of residues). Incertain embodiments, the Fc region-comprising antibody variant withimproved FcRn binding comprises amino acid substitutions at one, two orthree of positions 307, 380 and 434 of the Fc region thereof (Eunumbering of residues).

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In certain embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for antigen binding. Fora review of scFv see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994). HER2 antibody scFv fragments are described inWO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable regions thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). In certain embodiments,affinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity matured antibodies areproduced by procedures known in the art. Marks et al. Bio/Technology10:779-783 (1992) describes affinity maturation by VH and VL domainshuffling. Random mutagenesis of CDR and/or framework residues isdescribed by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813(1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

An “amino acid sequence variant” antibody herein is an antibody with anamino acid sequence which differs from a main species antibody. Incertain embodiments, amino acid sequence variants will possess at leastabout 70% homology with the main species antibody, or they will be atleast about 80%, or at least about 90% homologous with the main speciesantibody. The amino acid sequence variants possess substitutions,deletions, and/or additions at certain positions within or adjacent tothe amino acid sequence of the main species antibody. Examples of aminoacid sequence variants herein include an acidic variant (e.g.,deamidated antibody variant), a basic variant, an antibody with anamino-terminal leader extension (e.g. VHS-) on one or two light chainsthereof, an antibody with a C-terminal lysine residue on one or twoheavy chains thereof, etc, and includes combinations of variations tothe amino acid sequences of heavy and/or light chains. The antibodyvariant of particular interest herein is the antibody comprising anamino-terminal leader extension on one or two light chains thereof,optionally further comprising other amino acid sequence and/orglycosylation differences relative to the main species antibody.

A “glycosylation variant” antibody herein is an antibody with one ormore carbohydrate moieties attached thereto which differ from one ormore carbohydrate moieties attached to a main species antibody. Examplesof glycosylation variants herein include antibody with a G1 or G2oligosaccharide structure, instead a G0 oligosaccharide structure,attached to an Fc region thereof, antibody with one or two carbohydratemoieties attached to one or two light chains thereof, antibody with nocarbohydrate attached to one or two heavy chains of the antibody, etc,and combinations of glycosylation alterations. Where the antibody has anFc region, an oligosaccharide structure may be attached to one or twoheavy chains of the antibody, e.g. at residue 299 (298, Eu numbering ofresidues).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “immunosuppressive agent” as used herein for adjunct therapyrefers to substances that act to suppress or mask the immune system ofthe subject being treated herein. This would include substances thatsuppress cytokine production, down-regulate or suppress self-antigenexpression, or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir; tacrolimus;glucocorticoids such as cortisol or aldosterone; anti-inflammatoryagents such as a cyclooxygenase inhibitor; a 5-lipoxygenase inhibitor;or a leukotriene receptor antagonist; purine antagonists such asazathioprine or mycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporine; 6 mercaptopurine; steroids such as corticosteroids orglucocorticosteroids or glucocorticoid analogs, e.g., prednisone,methylprednisolone, including SOLU-MEDROL® methylprednisolone sodiumsuccinate, and dexamethasone; dihydrofolate reductase inhibitors such asmethotrexate (oral or subcutaneous); anti-malarial agents such aschloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokineor cytokine receptor antibodies or antagonists includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosisfactor (TNF)-alpha antibodies (infliximab (REMICADE®) or adalimumab),anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies,anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies,and anti-interleukin-6 (IL-6) receptor antibodies and antagonists;anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies;anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptidecontaining a LFA-3 binding domain (WO 90/08187 published Jul. 26, 1990);streptokinase; transforming growth factor-beta (TGF-beta);streptodomase; RNA or DNA from the host; FK506; RS-61443; chlorambucil;deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No.5,114,721); T-cell receptor fragments (Offner et al., Science, 251:430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO91/01133); BAFF antagonists such as BAFF or BR3 antibodies orimmunoadhesins and zTNF4 antagonists (for review, see Mackay and Mackay,Trends Immunol., 23:113-5 (2002) and see also definition below);biologic agents that interfere with T cell helper signals, such asanti-CD40 receptor or anti-CD40 ligand (CD154), including blockingantibodies to CD40-CD40 ligand. (e.g., Durie et al., Science, 261:1328-30 (1993); Mohan et al., J. Immunol., 154: 1470-80 (1995)) andCTLA4-Ig (Finck et al., Science, 265: 1225-7 (1994)); and T-cellreceptor antibodies (EP 340,109) such as T10B9.

The term “ameliorates” or “amelioration” as used herein refers to adecrease, reduction or elimination of a condition, disease, disorder, orphenotype, including an abnormality or symptom.

A “symptom” of a disease or disorder (e.g., inflammatory bowel disease,e.g., ulcerative colitis or Crohn's disease) is any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by a subject and indicative of disease.

The expression “therapeutically effective amount” refers to an amountthat is effective for preventing, ameliorating, or treating a disease ordisorder (e.g., inflammatory bowel disease, e.g., ulcerative colitis orCrohn's disease). For example, a “therapeutically effective amount” ofan antibody refers to an amount of the antibody that is effective forpreventing, ameliorating, or treating the specified disease or disorder.Similarly, a “therapeutically effective amount” of a combination of anantibody and a second compound refers to an amount of the antibody andan amount of the second compound that, in combination, is effective forpreventing, ameliorating, or treating the specified disease or disorder.

It is to be understood that the terminology “a combination of” twocompounds does not mean that the compounds have to be administered inadmixture with each other. Thus, treatment with or use of such acombination encompasses a mixture of the compounds or separateadministration of the compounds, and includes administration on the sameday or different days. Thus the terminology “combination” means two ormore compounds are used for the treatment, either individually or inadmixture with each other. When an antibody and a second compound, forexample, are administered in combination to a subject, the antibody ispresent in the subject at a time when the second compound is alsopresent in the subject, whether the antibody and second compound areadministered individually or in admixture to the subject. In certainembodiments, a compound other than the antibody is administered prior tothe antibody. In certain embodiments, a compound other than the antibodyis administered after the antibody.

For the purposes herein, “tumor necrosis factor-alpha (TNF-alpha)”refers to a human TNF-alpha molecule comprising the amino acid sequenceas described in Pennica et al., Nature, 312:721 (1984) or Aggarwal etal., JBC, 260:2345 (1985).

A “TNF-alpha inhibitor” herein is an agent that inhibits, to someextent, a biological function of TNF-alpha, generally through binding toTNF-alpha and neutralizing its activity. Examples of TNF inhibitorsspecifically contemplated herein are etanercept (ENBREL®), infliximab(REMICADE®), adalimumab (HUMTRA®), golimumab (SIMPONI™), andcertolizumab pegol (CIMZIA®).

“Corticosteroid” refers to any one of several synthetic or naturallyoccurring substances with the general chemical structure of steroidsthat mimic or augment the effects of the naturally occurringcorticosteroids. Examples of synthetic corticosteroids includeprednisone, prednisolone (including methylprednisolone), dexamethasonetriamcinolone, and betamethasone.

An “antagonist” refers to a molecule capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with the activities of aparticular or specified protein, including its binding to one or morereceptors in the case of a ligand or binding to one or more ligands incase of a receptor. Antagonists include antibodies and antigen-bindingfragments thereof, proteins, peptides, glycoproteins, glycopeptides,glycolipids, polysaccharides, oligosaccharides, nucleic acids,bioorganic molecules, peptidomimetics, pharmacological agents and theirmetabolites, transcriptional and translation control sequences, and thelike. Antagonists also include small molecule inhibitors of the protein,and fusion proteins, receptor molecules and derivatives which bindspecifically to the protein thereby sequestering its binding to itstarget, antagonist variants of the protein, antisense molecules directedto the protein, RNA aptamers, and ribozymes against the protein.

A “self-inject device” refers to a medical device forself-administration, e.g., by a patient or in-home caregiver, of atherapeutic agent. Self-inject devices include autoinjector devices andother devices designed for self-administration.

“Oligonucleotide,” as used herein, refers to short, single strandedpolynucleotides that are at least about seven nucleotides in length andless than about 250 nucleotides in length. Oligonucleotides may besynthetic. The terms “oligonucleotide” and “polynucleotide” are notmutually exclusive. The description above for polynucleotides is equallyand fully applicable to oligonucleotides.

The term “primer” refers to a single stranded polynucleotide that iscapable of hybridizing to a nucleic acid and allowing the polymerizationof a complementary nucleic acid, generally by providing a free 3′-OHgroup.

The term “amplification” refers to the process of producing one or morecopies of a reference nucleic acid sequence or its complement.Amplification may be linear or exponential (e.g., PCR). A “copy” doesnot necessarily mean perfect sequence complementarity or identityrelative to the template sequence. For example, copies can includenucleotide analogs such as deoxyinosine, intentional sequencealterations (such as sequence alterations introduced through a primercomprising a sequence that is hybridizable, but not fully complementary,to the template), and/or sequence errors that occur duringamplification.

The term “detection” includes any means of detecting, including directand indirect detection.

“Elevated expression” or “elevated levels” refers to an increasedexpression of a mRNA or a protein in a patient relative to a control,such as an individual or individuals who are not suffering from anautoimmune disease, e.g., IBD, or relative to a pre-establishedthreshold or cut-off value, or relative to the median for a populationof patients and/or subjects.

“Low expression” or “low expression levels” refers to a decreasedexpression of a mRNA or a protein in a patient relative to a control,such as an individual or individuals who are not suffering from anautoimmune disease, e.g., IBD, or relative to a pre-establishedthreshold or cut-off value, or relative to the median for a populationof patients and/or subjects.

The term “multiplex-PCR” refers to a single PCR reaction carried out onnucleic acid obtained from a single source (e.g., a patient) using morethan one primer set for the purpose of amplifying two or more DNAsequences in a single reaction.

The term “biomarker” as used herein refers to an indicator of aphenotype of a patient, e.g., a pathological state or likelyresponsiveness to a therapeutic agent, which can be detected in abiological sample of the patient. Biomarkers include, but are notlimited to, DNA, RNA, protein, carbohydrate, or glycolipid-basedmolecular markers.

The term “diagnosis” is used herein to refer to the identification orclassification of a molecular or pathological state, disease orcondition. For example, “diagnosis” may refer to identification of aparticular type of IBD, e.g., UC or Crohn's disease. “Diagnosis” mayalso refer to the classification of a particular subtype of IBD, e.g.,by histopathological criteria or by molecular features (e.g., a subtypecharacterized by expression of one or a combination of particular genesor proteins encoded by said genes).

The term “aiding diagnosis” is used herein to refer to methods thatassist in making a clinical determination regarding the presence, ornature, of a particular type of symptom or condition. For example, amethod of aiding diagnosis of IBD can comprise measuring the expressionof certain genes in a biological sample from an individual.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of autoimmune disorder-attributable disease symptoms of anautoimmune disease such as IBD.

The term “prediction” is used herein to refer to the likelihood that apatient will respond either favorably or unfavorably to a drug(therapeutic agent) or set of drugs or a therapeutic regimen. In oneembodiment, the prediction relates to the extent of those responses. Inone embodiment, the prediction relates to whether and/or the probabilitythat a patient will survive or improve following treatment, for exampletreatment with a particular therapeutic agent, or for a certain periodof time without disease recurrence. The predictive methods of theinvention can be used clinically to make treatment decisions by choosingthe most appropriate treatment modalities for any particular patient.The predictive methods of the present invention are valuable tools inpredicting if a patient is likely to respond favorably to a treatmentregimen, such as a given therapeutic regimen, including for example,administration of a given therapeutic agent or combination, surgicalintervention, steroid treatment, etc., or whether long-term survival ofthe patient or remission or sustained remission, following a therapeuticregimen is likely.

A “control subject” refers to a healthy subject who has not beendiagnosed as having a particular disease, e.g., IBD, and who does notsuffer from any sign or symptom associated with that disease.

By “correlate” or “correlating” is meant comparing, in any way, theperformance and/or results of a first analysis or protocol with theperformance and/or results of a second analysis or protocol. Forexample, one may use the results of a first analysis or protocol incarrying out a second protocols and/or one may use the results of afirst analysis or protocol to determine whether a second analysis orprotocol should be performed. With respect to the embodiment of geneexpression analysis or protocol, one may use the results of the geneexpression analysis or protocol to determine whether a specifictherapeutic regimen should be performed.

The term “comparing” as used herein refers to comparing the level of thebiomarker in the sample from the individual or patient with thereference level of the biomarker specified elsewhere in thisdescription. It is to be understood that comparing as used hereinusually refers to a comparison of corresponding parameters or values,e.g., an absolute amount is compared to an absolute reference amountwhile a concentration is compared to a reference concentration or anintensity signal obtained from the biomarker in a sample is compared tothe same type of intensity signal obtained from a reference sample. Thecomparison may be carried out manually or computer assisted. Thus, thecomparison may be carried out by a computing device (e.g., of a systemdisclosed herein). The value of the measured or detected level of thebiomarker in the sample from the individual or patient and the referencelevel can be, e.g., compared to each other and the said comparison canbe automatically carried out by a computer program executing analgorithm for the comparison. The computer program carrying out the saidevaluation will provide the desired assessment in a suitable outputformat. For a computer assisted comparison, the value of the determinedamount may be compared to values corresponding to suitable referenceswhich are stored in a database by a computer program. The computerprogram may further evaluate the result of the comparison, i.e.automatically provide the desired assessment in a suitable outputformat. For a computer assisted comparison, the value of the determinedamount may be compared to values corresponding to suitable referenceswhich are stored in a database by a computer program. The computerprogram may further evaluate the result of the comparison, i.e.automatically provides the desired assessment in a suitable outputformat.

The phrase “recommending a treatment” as used herein refers to using theinformation or data generated relating to the level or presence ofintegrin beta7 mRNA or protein, integrin alphaE mRNA or protein, orCD3epsilon mRNA or protein in a sample of a patient to identify thepatient as suitably treated or not suitably treated with a therapy. Insome embodiment the therapy may comprise an integrin beta7 antagonist,including an anti-integrin beta7 antibody such as etrolizumab. In someembodiments the phrase “recommending a treatment/therapy” includes theidentification of a patient who requires adaptation of an effectiveamount of the integrin beta7 antagonist being administered. In someembodiments recommending a treatment includes recommending that theamount of integrin beta7 antagonist being administered is adapted. Thephrase “recommending a treatment” as used herein also may refer to usingthe information or data generated for proposing or selecting a therapycomprising an integrin beta7 antagonist for a patient identified orselected as more or less likely to respond to the therapy comprising anintegrin beta7 antagonist. The information or data used or generated maybe in any form, written, oral or electronic. In some embodiments, usingthe information or data generated includes communicating, presenting,reporting, storing, sending, transferring, supplying, transmitting,dispensing, or combinations thereof. In some embodiments, communicating,presenting, reporting, storing, sending, transferring, supplying,transmitting, dispensing, or combinations thereof are performed by acomputing device, analyzer unit or combination thereof. In some furtherembodiments, communicating, presenting, reporting, storing, sending,transferring, supplying, transmitting, dispensing, or combinationsthereof are performed by a laboratory or medical professional. In someembodiments, the information or data includes a comparison of the levelof integrin beta7 mRNA or protein, integrin alphaE mRNA or protein, orCD3epsilon mRNA or protein to a reference level. In some embodiments,the information or data includes an indication that integrin beta7 mRNAor protein, integrin alphaE mRNA or protein, or CD3epsilon mRNA orprotein is present or absent in the sample. In some embodiments, theinformation or data includes an indication that the patient is suitablytreated or not suitably treated with a therapy comprising an integrinbeta7 antagonist, including an anti-integrin beta7 antibody such asetrolizumab.

A “package insert” is used to refer to instructions customarily includedin commercial packages of therapeutic products or medicaments, thatcontain information about the indications, usage, dosage,administration, contraindications, other therapeutic products to becombined with the packaged product, and/or warnings concerning the useof such therapeutic products or medicaments and the like.

A “kit” is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g., a medicament for treatment of an IBD, e.g., UCor Crohn's disease, or a probe for specifically detecting a biomarkergene or protein of the invention. In certain embodiments, themanufacture is promoted, distributed, or sold as a unit for performingthe methods of the present invention.

A “target audience” is a group of people or an institution to whom or towhich a particular medicament is being promoted or intended to bepromoted, as by marketing or advertising, especially for particularuses, treatments, or indications, such as individual patients, patientpopulations, readers of newspapers, medical literature, and magazines,television or internet viewers, radio or internet listeners, physicians,drug companies, etc.

The term “serum sample” refers to any serum sample obtained from anindividual. Methods for obtaining sera from mammals are well known inthe art.

The term “whole blood” refers to any whole blood sample obtained from anindividual. Typically, whole blood contains all of the blood components,e.g., cellular components and plasma. Methods for obtaining whole bloodfrom mammals are well known in the art.

The expression “not responsive to,” “non-response” and grammaticalvariants thereof, as it relates to the reaction of subjects or patientsto one or more of the medicaments (therapeutic agents) that werepreviously administered to them, describes those subjects or patientswho, upon administration of such medicament(s), did not exhibit any oradequate signs of treatment of the disorder for which they were beingtreated, or they exhibited a clinically unacceptably high degree oftoxicity to the medicament(s), or they did not maintain the signs oftreatment after first being administered such medicament(s), with theword treatment being used in this context as defined herein. The phrase“not responsive” includes a description of those subjects who areresistant and/or refractory to the previously administeredmedication(s), and includes the situations in which a subject or patienthas progressed while receiving the medicament(s) that he or she is beinggiven, and in which a subject or patient has progressed within 12 months(for example, within six months) after completing a regimen involvingthe medicament(s) to which he or she is no longer responsive. Thenon-responsiveness to one or more medicaments thus includes subjects whocontinue to have active disease following previous or current treatmenttherewith. For instance, a patient may have active disease activityafter about one to three months, or three to six months, or six to 12months, of therapy with the medicament(s) to which they arenon-responsive. Such responsiveness may be assessed by a clinicianskilled in treating the disorder in question.

For purposes of non-response to medicament(s), a subject who experiences“a clinically unacceptably high level of toxicity” from previous orcurrent treatment with one or more medicaments experiences one or morenegative side-effects or adverse events associated therewith that areconsidered by an experienced clinician to be significant, such as, forexample, serious infections, congestive heart failure, demyclination(leading to multiple sclerosis), significant hypersensitivity,neuropathological events, high degrees of autoimmunity, a cancer such asendometrial cancer, non-Hodgkin's lymphoma, breast cancer, prostatecancer, lung cancer, ovarian cancer, or melanoma, tuberculosis (TB), andthe like.

The “amount” or “level” of a biomarker associated with an increasedclinical benefit to a patient suffering from a certain disease ordisorder, or predictive of response to a particular therapeutic agent ortreatment regimen, is a detectable level in a biological sample. Thesecan be measured by methods known to one skilled in the art and alsodisclosed herein. The expression level or amount of biomarker assessedcan be used to determine the response or the predicted response to atreatment or therapeutic agent.

The terms “level of expression” or “expression level” in general areused interchangeably and generally refer to the amount of apolynucleotide or an amino acid product or protein in a biologicalsample. “Expression” generally refers to the process by whichgene-encoded information is converted into the structures present andoperating in the cell. Therefore, as used herein, “expression” of a genemay refer to transcription into a polynucleotide, translation into aprotein, or even posttranslational modification of the protein.Fragments of the transcribed polynucleotide, the translated protein, orthe post-translationally modified protein shall also be regarded asexpressed whether they originate from a transcript generated byalternative splicing or a degraded transcript, or from aposttranslational processing of the protein, e.g., by proteolysis.“Expressed genes” include those that are transcribed into apolynucleotide as mRNA and then translated into a protein, and alsothose that are transcribed into RNA but not translated into a protein(for example, transfer and ribosomal RNAs).

A variety of additional terms are defined or otherwise characterizedherein.

Compositions and Methods

A. Beta7 Integrin Antagonists

Methods of treating a gastrointestinal inflammatory disorder in asubject, e.g., a human, by administering beta7 integrin antagonists areprovided. Examples of potential antagonists include an oligonucleotidethat binds to the fusions of immunoglobulin with beta7 integrin, and, inparticular, antibodies including, without limitation, poly- andmonoclonal antibodies and antibody fragments, single-chain antibodies,anti-idiotypic antibodies, and chimeric or humanized versions of suchantibodies or fragments, as well as human antibodies and antibodyfragments. Alternatively, a potential antagonist may be a closelyrelated protein, for example, a mutated form of the beta7 integrin thatrecognizes the ligand but imparts no effect, thereby competitivelyinhibiting the action of the beta7 integrin.

Another potential beta7 integrin antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes thebeta7 integrin herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the beta7 integrin. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into beta7 integrin protein (antisense—Okano, Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression (CRC Press: Boca Raton, Fla., 1988). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of the PROpolypeptide. When antisense DNA is used, oligodeoxyribonucleotidesderived from the translation-initiation site, e.g., between about −10and +10 positions of the target gene nucleotide sequence, are typical.

Other potential antagonists include small molecules that bind to theactive site, the ligand or binding molecule binding site, therebyblocking the normal biological activity of the beta7 integrin. Examplesof small molecules include, but are not limited to, small peptides orpeptide-like molecules, typically soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can-be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT Publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT Publication No. WO 97/33551. These small molecules can beidentified by any one or more of the screening assays discussedhereinabove and/or by any other screening techniques well known forthose skilled in the art.

Screening assays for antagonists are designed to identify compounds thatbind or complex with the beta7 integrin encoded by the genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

B. Anti-Beta7 Integrin Antibodies

In one embodiment, the beta7 integrin antagonists are anti-beta7antibodies. Exemplary antibodies include polyclonal, monoclonal,humanized, human, bispecific, and heteroconjugate antibodies, etc., asdescribed below.

1. Polyclonal Antibodies

Polyclonal antibodies can be raised in animals by multiple subcutaneous(SC) or intraperitoneal (IP) injections of the relevant antigen and anadjuvant. It may be useful to conjugate the relevant antigen to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. In certain embodiments, the animalis boosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium may contain one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells(also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

In certain embodiments, fusion partner myeloma cells are those that fuseefficiently, support stable high-level production of antibody by theselected antibody-producing cells, and are sensitive to a selectivemedium that selects against the unfused parental cells. In certainembodiments, myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 andderivatives e.g., X63-Ag8-653 cells available from the American TypeCulture Collection, Manassas, Va., USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); andBrodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen. Incertain embodiments, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980). Once hybridoma cells that produce antibodiesof the desired specificity, affinity, and/or activity are identified,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)). Suitable culture media forthis purpose include, for example, DMEM or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal e.g., by i.p. injection of the cells into mice. The monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional antibodypurification procedures such as, for example, affinity chromatography(e.g., using protein A or protein G-Sepharose) or ion-exchangechromatography, hydroxylapatite chromatography, gel electrophoresis,dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as asource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myelomacells that do not otherwise produce antibody protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of DNA encoding theantibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using e.g., thetechniques described in McCafferty et al., Nature, 348:552-554 (1990).Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (CH and CL) sequences for thehomologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Exemplary anti-beta7 antibodies are Fib504, Fib 21, 22, 27, 30(Tidswell, M. J Immunol. 1997 Aug. 1; 159(3):1497-505) or humanizedderivatives thereof. Humanized antibodies of Fib504 was disclosed indetail in U.S. Patent Publication No. 20060093601 (issued as U.S. Pat.No. 7,528,236), the content of which is incorporated by reference in itsentirety (also see discussion below).

3. Human and Humanized Antibodies

The anti-beta7 integrin antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies. The choice of human variable domains, bothlight and heavy, to be used in making the humanized antibodies is veryimportant to reduce antigenicity and HAMA response (human anti-mouseantibody) when the antibody is intended for human therapeutic use.According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human V domainsequence which is closest to that of the rodent is identified and thehuman framework region (FR) within it accepted for the humanizedantibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J.Mol. Biol., 196:901 (1987)). Another method uses a particular frameworkregion derived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chains. The same framework may beused for several different humanized antibodies (Carter et al., Proc.Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993)). It is further important that antibodies be humanizedwith retention of high binding affinity for the antigen and otherfavorable biological properties. To achieve this goal, according tocertain embodiments, humanized antibodies are prepared by a process ofanalysis of the parental sequences and various conceptual humanizedproducts using three-dimensional models of the parental and humanizedsequences. Three-dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

Various forms of a humanized Anti-beta7 integrin antibody arecontemplated. For example, the humanized antibody may be an antibodyfragment, such as a Fab, which is optionally conjugated with one or morecytotoxic agent(s) in order to generate an immunoconjugate.Alternatively, the humanized antibody may be an intact antibody, such asan intact IgG1 antibody.

Exemplary humanized anti-beta7 antibodies include, but are not limitedto rhuMAb Beta7, which is a humanized monoclonal antibody against theintegrin subunit 37 and was derived from the rat anti-mouse/humanmonoclonal antibody FIB504 (Andrew et al., 1994 J Immunol 1994;153:3847-61). It has been engineered to include human immunoglobulinIgG1 heavy chain and κ1 light chain frameworks and is produced byChinese hamster ovary cells. This antibody binds to two integrins, α4β7(Holzmann et al. 1989 Cell, 1989; 56:37-46; Hu et al., 1992, Proc NatlAcad Sci USA 1992; 89:8254-8) and αEβ7 (Cepek et al., 1993 J Immunol1993; 150:3459-70), which regulate trafficking and retention oflymphocyte subsets in the gastrointestinal tract and are involved ininflammatory bowel diseases (IBD) such as ulcerative colitis (UC) andCrohn's disease (CD). rhuMAb Beta7 is a potent in vitro blocker of thecellular interaction between α4β7 and its ligands (mucosal addressincell adhesion molecule-1 [MAdCAM]-1, vascular cell adhesion molecule[VCAM]-1, and fibronectin) as well as the interaction between αEβ7 andits ligand (E-cadherin). rhuMAb Beta7 binds reversibly, with similarhigh affinity, to β7 on lymphocytes from rabbits, cynomolgus monkeys,and humans. It also binds to mouse β7 with high affinity. The amino acidsequence as well as the making and using of rhuMAb Beta7 and itsvariants are disclosed in detail in e.g., U.S. Patent ApplicationPublication No. 20060093601 (issued as U.S. Pat. No. 7,528,236), thecontent of which is incorporated in its entirety.

FIGS. 1A and 1B depict alignment of sequences of the variable light andheavy chains for the following: light chain human subgroup kappa Iconsensus sequence (FIG. 1A, SEQ ID NO:12), heavy chain human subgroupIII consensus sequence (FIG. 1B, SEQ ID NO:13), rat anti-mouse beta7antibody (Fib504) variable light chain (FIG. 1A, SEQ ID NO:10), ratanti-mouse beta7 antibody (Fib504) variable heavy chain (FIG. 1B, SEQ IDNO: 11), and humanized antibody variants: Humanized hu504Kgraft variablelight chain (FIG. 1A, SEQ ID NO:14), humanized hu504K graft variableheavy chain (FIG. 1B, SEQ ID NO:15), variants hu504-5, hu504-16, andhu504-32 (amino acid variations from humanized hu504K graft areindicated in FIG. 1A (light chain) (SEQ ID NOS:22-24, respectively, inorder of appearance) and FIG. 1B (heavy chain) for variants hu504-5,hu504-16, and 504-32 (SEQ ID NO:25).

4. Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

5. Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody,” e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

6. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of beta7 integrin as described herein.Other such antibodies may combine a TAT binding site with a binding sitefor another protein. Alternatively, an anti-Beta7 integrin arm may becombined with an arm which binds to a triggering molecule on a leukocytesuch as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG(Fc.γ.R), such as Fc.γRI (CD64), Fc.γRII (CD32) and Fc.γ.RIII (CD16), soas to focus and localize cellular defense mechanisms to theTAT-expressing cell. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express TAT. These antibodies possess aTAT-binding arm and an arm which binds the cytotoxic agent (e.g.,saporin, anti-interferon-.alpha., vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.,F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. In certainembodiments, the fusion is with an Ig heavy chain constant domain,comprising at least part of the hinge, C_(H2), and C_(H3) regions. Incertain embodiments, the first heavy-chain constant region (C_(H1))containing the site necessary for light chain bonding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost cell. This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In certain embodiments, the bispecific antibodies are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. In certain embodiments, the interfacecomprises at least a part of the C_(H3) domain. In this method, one ormore small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′).sub.2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives: One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. Various techniques for making and isolatingbispecific antibody fragments directly from recombinant cell culturehave also been described. For example, bispecific antibodies have beenproduced using leucine zippers. Kostelny et al., J. Immunol.148(5):1547-1553 (1992). The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a V.sub.H connected to a V.sub.L by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V.sub.H and V.sub.L domains of one fragment are forcedto pair with the complementary V.sub.L and V.sub.H domains of anotherfragment, thereby forming two antigen-binding sites. Another strategyfor making bispecific antibody fragments by the use of single-chain Fv(sFv) dimers has also been reported. Seec Gruber et al., J. Immunol.,152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

7. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

8. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g., tetravalent antibodies),which can be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. In certain embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In certain embodiments, themultivalent antibody herein comprises (or consists of) three to abouteight, but typically four, antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (and typically twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a firstvariable domain, VD2 is a second variable domain, Fc is one polypeptidechain of an Fc region, X1 and X2 represent an amino acid or polypeptide,and n is 0 or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (and typically four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

9. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

10. Immunoconjugates

The antagonist or antibody used in the methods herein is optionallyconjugated to another agent, such as a cytotoxic agent, or cytokine.

Conjugation will ordinarily be achieved through a covalent linkage, theprecise nature of which will be determined by the targeting molecule andthe linking site on the integrin beta7 antagonist or antibodypolypeptide. Typically, a non-peptidic agent is modified by the additionof a linker that allows conjugation to anti-beta7 integrin antibodythrough its amino acid side chains, carbohydrate chains, or reactivegroups introduced on antibody by chemical modification. For example, adrug may be attached through the .epsilon.-amino group of a lysineresidue, through a free .alpha.-amino group, by disulfide exchange to acysteine residue, or by oxidation of the 1,2-diols in a carbohydratechain with periodic acid to allow attachment of drugs containing variousnucleophiles through a Schiff-base linkage. See, for example, U.S. Pat.No. 4,256,833. Protein modifying agents include amine-reactive reagents(e.g., reactive esters, isothiocyanates, aldehydes, and sulfonylhalides), thiol-reactive reagents (e.g., haloacetyl derivatives andmaleimides), and carboxylic acid- and aldehyde-reactive reagents.Integrin beta7 antagonist or antibody polypeptides can be covalentlyjoined to peptidic agents through the use of bifunctional cross-linkingreagents. Heterobifunctional reagents are more commonly used and permitthe controlled coupling of two different proteins through the use of twodifferent reactive moieties (e.g., amine-reactive plus thiol,iodoacetamide, or maleimide). The use of such linking agents is wellknown in the art. See, for example, Brinkley, supra, and U.S. Pat. No.4,671,958. Peptidic linkers can also be employed. In the alternative, ananti-beta7 integrin antibody polypeptide can be linked to a peptidicmoiety through preparation of a fusion polypeptide.

Examples of further bifunctional protein coupling agents includeN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

11. Immunoliposomes

The anti-beta7 integrin antibodies disclosed herein may also beformulated as immunoliposomes. A “liposome” is a small vesicle composedof various types of lipids, phospholipids and/or surfactant which isuseful for delivery of a drug to a mammal. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes. Liposomes containing theantibody are prepared by methods known in the art, such as described inEpstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al.,Proc. Natl Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

12. Vectors, Host Cells and Recombinant Methods for Antibody Production

Also provided are isolated nucleic acids encoding the anti-beta7antibodies or polypeptide agents described herein, vectors and hostcells comprising the nucleic acids and recombinant techniques for theproduction of the antibodies.

For recombinant production of the antibody, the nucleic acid encoding itmay be isolated and inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. In anotherembodiment, the antibody may be produced by homologous recombination,e.g., as described in U.S. Pat. No. 5,204,244, specifically incorporatedherein by reference. DNA encoding the monoclonal antibody is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, e.g., as described in U.S.Pat. No. 5,534,615 issued Jul. 9, 1996 and specifically incorporatedherein by reference.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coli X1776(ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. Theseexamples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-beta7integrin antibody-encoding vectors. Saccharomyces cerevisiae, or commonbaker's yeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-Beta7antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-beta7 integrin antibody production and culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce the anti-beta7 integrin antibody of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham et al., Meth. Enz. 58:44 (1979),Barnes et al., Anal. Biochem. 1 02:255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195;or U.S. Patent Re. 30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the typical purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human .gamma.1, .gamma.2,or .gamma.4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13(1983)). Protein G is recommended for all mouse isotypes and for human.gamma.3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C.sub.H3 domain, the Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Othertechniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered. Following any preliminary purification step(s), the mixturecomprising the antibody of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, typically performed at low saltconcentrations (e.g., from about 0-0.25 M salt).

C. Pharmaceutical Formulations

Therapeutic formulations comprising the therapeutic agents, antagonistsor antibodies of the invention are prepared for storage by mixing theantibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of aqueous solutions, lyophilized or other driedformulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, typically thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and .gamma. ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

D. Administration

The physician administering treatment will be able to determine theappropriate dose for the individual subject for weight-based dosing or,for flat dosing, will follow the instructions on the label. Preparationand dosing schedules for commercially available second therapeutic andother compounds administered in combination with the integrin beta7antagonists may be used according to manufacturers' instructions ordetermined empirically by the skilled practitioner.

For the prevention or treatment of disease, the appropriate dosage ofthe integrin beta7 antagonist and any second therapeutic or othercompound administered in combination with the non-depleting antibodywill depend on the type of gastrointestinal inflammatory disorder to betreated, e.g., IBD, UC, CD, the severity and course of the disease,whether the integrin beta7 antagonist or combination is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the integrin beta7 antagonist orcombination, and the discretion of the attending physician. The integrinbeta7 antagonist or combination is suitably administered to the patientat one time or more typically over a series of treatments. In certainembodiments, the integrin beta7 antagonist is administered once everyweek, or once every two weeks, or once every four weeks, or once everysix weeks, or once every eight weeks for a period of one month (4weeks), or two months, three months, or six months, or 12 months, or 18months, or 24 months, or chronically for the lifetime of the patient. Incertain embodiments, the treatment is self-administered by the patient.

Depending on the type and severity of the disease, about 0.5 mg/kg to4.0 mg/kg of anti-beta7 antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful.

For example, in certain embodiments, a flat dose of anti-beta7 antibodyis administered to the patient. A flat dose is a particular amount ofanti-beta7 antibody that is administered to every patient regardless ofweight. Depending on the type and severity of the disease, a flat doseof between about 50 mg and 450 mg of anti-beta7 antibody is administeredto the patient, which may be one or more separate injections orinfusions or administrations. Such flat dose can be administeredintravenously or subcutaneously or by other routes as described herein.In certain embodiments, the flat dose is 50 mg, or 100 mg, or 150 mg, or200 mg, or 300 mg, or 400 mg, or 420 mg, or 450 mg.

In certain embodiments, an initial flat loading dose of anti-beta7antibody is followed by one or more flat maintenance doses of anti-beta7antibody. The loading dose is a larger quantity of anti-beta7 antibodythan the maintenance dose. In certain embodiments, the loading dose isbetween about 400 mg and 450 mg and the maintenance dose is betweenabout 50 mg and 350 mg. In certain embodiments, the loading dose is 400mg, or 420 mg, or 430 mg, or 450 mg. In certain embodiments, themaintenance dose is 50 mg, or 100 mg, or 150 mg, or 200 mg, or 300 mg,or 350 mg.

Typically, the clinician will administer an antibody (alone or incombination with a second compound) of the invention until a dosage(s)is reached that provides the required biological effect. The progress ofthe therapy of the invention is easily monitored by conventionaltechniques and assays.

The integrin beta7 antagonist can be administered by any suitable means,including parenteral, topical, intravenous, subcutaneous,intraperitoneal, intrapulmonary, intranasal, and/or intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration.Intrathecal administration is also contemplated (see, e.g., U.S. PatentPublication No. 2002/0009444 by Grillo-Lopez). In addition, the integrinbeta7 antagonist may suitably be administered by pulse infusion, e.g.,with declining doses of the antibody. In certain embodiments, the dosingis given intravenously or subcutaneously. Each exposure may be providedusing the same or a different administration means. In one embodiment,each exposure to anti-beta7 antibody is by subcutaneous administration.In one embodiment, the first exposure to anti-beta7 antibody, e.g., theloading dose, is by intravenous administration and each subsequentexposure is by subcutaneous administration.

In certain embodiments, an anti-beta7 antibody is administered using,for example, a self-inject device, autoinjector device, or other devicedesigned for self-administration. Various self-inject devices, includingautoinjector devices, are known in the art and are commerciallyavailable. Exemplary devices include, but are not limited to, prefilledsyringes (such as BD HYPAK SCF®, READYFILL™, and STERIFILL SCF™ fromBecton Dickinson; CLEARSHOT™ copolymer prefilled syringes from Baxter;and Daikyo Seiko CRYSTAL ZENITH® prefilled syringes available from WestPharmaceutical Services); disposable pen injection devices such as BDPen from Becton Dickinson; ultra-sharp and microneedle devices (such asINJECT-EASE™ and microinfuser devices from Becton Dickinson; andH-PATCH™ available from Valeritas) as well as needle-free injectiondevices (such as BIOJECTOR® and IJECT® available from Bioject; andSOF-SERTER® and patch devices available from Medtronic). In certainembodiments, rhuMAb Beta7 is an article of manufacture comprising aprefilled syringe comprising 2 ML (150 mg) rhuMAb Beta7. In certainembodiments, rhuMAb Beta7 is an article of manufacture comprising aprefilled syringe comprising 1 ML (180 mg) rhuMAb Beta7.

As noted, the integrin beta7 antagonist can be administered alone or incombination with at least a second therapeutic compound. These secondtherapeutic compounds are generally used in the same dosages and withadministration routes as used heretofore, or about from 1 to 99% of theheretofore-employed dosages. If such second compounds are used, they areused in certain embodiments in lower amounts than if the integrin beta7antagonist were not present, so as to eliminate or reduce side effectscaused thereby.

Also as noted (e.g., see below), a variety of suitable secondtherapeutic compounds for the treatment of IBD, e.g., ulcerative colitisand Crohn's disease are known in the art, and dosages and administrationmethods for such second therapeutic compounds have likewise beendescribed.

Administration of the integrin beta7 antagonist and any secondtherapeutic compound can be done simultaneously, e.g., as a singlecomposition or as two or more distinct compositions using the same ordifferent administration routes. Alternatively, or additionally, theadministration can be done sequentially, in any order. In certainembodiments, intervals ranging from minutes to days, to weeks to months,can be present between the administrations of the two or morecompositions. For example, the integrin beta7 antagonist may beadministered first, followed by the second therapeutic compound.However, simultaneous administration or administration of the secondtherapeutic compound prior to the integrin beta7 antagonist is alsocontemplated.

The standard of care for subjects with active moderate-severe active UCinvolves therapy with standard doses of: an aminosalicylate, an oralcorticosteroid, 6-mercaptopurine (6-MP) and/or azathioprine. Therapywith an integrin beta7 antagonist, such as an anti-beta7 integrinantibody as disclosed herein will result in an improvement in diseaseremission (rapid control of disease and/or prolonged remission), and/orclinical response, superior to that achieved with the standard of carefor such subjects.

In one embodiment, the treatment of the present invention forinflammatory bowel disease (IBD) in a human subject with IBD comprisesadministering to the subject an effective amount of an therapeuticagent, such as an anti-beta7 integrin antibody, and further comprisingadministering to the subject an effective amount of a second medicament,that is an immunosuppressant, a pain-control agent, an antidiarrhealagent, an antibiotic, or a combination thereof.

In an exemplary embodiment, said secondary medicine is selected from thegroup consisting of an aminosalicylate, an oral corticosteroid,6-mercaptopurine (6-MP) and azathioprine. In another exemplaryembodiment, said secondary medicine is another integrin beta7antagonist, such as another anti-beta7 integrin antibody or an antibodyagainst a cytokine.

All these second medicaments may be used in combination with each otheror by themselves with the first medicament, so that the expression“second medicament” as used herein does not mean it is the onlymedicament besides the first medicament, respectively. Thus, the secondmedicament need not be one medicament, but may constitute or comprisemore than one such drug.

Combined administration herein includes co-administration, usingseparate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein generally there is atime period while both (or all) active agents simultaneously exert theirbiological activities.

The combined administration of a second medicament includesco-administration (concurrent administration), using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein generally there is a time periodwhile both (or all) active agents (medicaments) simultaneously exerttheir biological activities.

E. Design Treatment Regimens

Drug development is a complex and expensive process. The cost ofbringing a new drug to market is estimated to be between $800 millionand $1 billion. Less than 10% of drugs in phase I clinical trials makeit to the approval phase. Two key reasons why drugs fail at late stagesare a lack of understanding of the relationship betweendose-concentration response and unanticipated safety events. Given thisscenario, it is important to have enabling tools that help predict how adrug will perform in vivo and assist in the success of a clinicaltherapeutic candidate (Lakshmi Kamath, Drug Discovery and Development;Modeling Success in PK/PD Testing Drug Discovery & Development (2006)).

Pharmacokinetics (PK) characterizes the absorption, distribution,metabolism, and elimination properties of a drug. Pharmacodynamics (PD)defines the physiological and biological response to the administereddrug. PK/PD modeling establishes a mathematical and theoretical linkbetween these two processes and helps better predict drug action.Integrated PK/PD modeling and computer-assisted trial design viasimulation are being incorporated into many drug development programsand are having a growing impact (Lakshmi Kamath, Drug Discovery andDevelopment; Modeling Success in PK/PD Testing Drug Discovery &Development (2006)).

PK/PD testing is typically performed at every stage of the drugdevelopment process. Because development is becoming increasinglycomplex, time consuming, and cost intensive, companies are looking tomake better use of PK/PD data to eliminate flawed candidates at thebeginning and identify those with the best chance of clinical success.(Lakshmi Kamath, supra).

PK/PD modeling approaches are proving useful in determiningrelationships between biomarker response, drug levels, and dosingregimens. The PK/PD profile of a drug candidate and the ability topredict a patient's response to it are critical to the success ofclinical trials. Recent advances in molecular biology techniques and abetter understanding of targets for various diseases have validatedbiomarkers as a good clinical indicator of a drug's therapeuticefficacy. Biomarker assays (including those described herein) and use ofsuch biomarker assays help identify a biological response to a drugcandidate. Once a biomarker is clinically validated, trial simulationscan be effectively modeled. Biomarkers have the potential to achievesurrogate status that may someday substitute for clinical outcomes indrug development. (Lakshmi Kamath, supra).

The amount of biomarkers in the peripheral blood such as those describedherein can be used in identifying the biological response to a treatmentwith integrin beta7 antagonists and can therefore function as a goodclinical indicator for the therapeutic efficacy of a candidatetreatment.

Traditional PK/PD modeling in drug development defines parameters suchas drug dose concentration, drug exposure effects, drug half-life, drugconcentrations against time, and drug effects against time. When usedmore broadly, quantitative techniques such as drug modeling, diseasemodeling, trial modeling, and market modeling can support the entiredevelopment process, which results in better decisions through explicitconsideration of risk and better utilization of knowledge. A variety ofPK/PD modeling tools are available to drug development researchers, forexample, WinNonlin and the Knowledgebase Server (PKS) developed byPharsight, Inc. Mountain View, Calif.

General Biomarker Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994).

Primers, oligonucleotides and polynucleotides employed in the presentinvention can be generated using standard techniques known in the art.

Gene expression biomarkers associated with predicting responsiveness ofIBD patients including patient suffering from UC or Crohn's disease tocertain therapeutic agents are provided herein. These expression levelsof the mRNA or individual proteins encoded by the genes constitutebiomarkers for predicting responsiveness to IBD therapeutic agents, UCtherapeutic agents, and/or Crohn's disease therapeutic agents.Accordingly, the invention disclosed herein is useful in a variety ofsettings, e.g., in methods and compositions related to diagnosis andtherapy of inflammatory bowel diseases.

Detection of Gene Expression Levels

Nucleic acid, according to any of the methods described herein may beRNA transcribed from genomic DNA or cDNA generated from RNA or mRNA.Nucleic acid may be derived from a vertebrate, e.g., a mammal. A nucleicacid is said to be “derived from” a particular source if it is obtaineddirectly from that source or if it is a copy of a nucleic acid found inthat source.

Nucleic acid includes copies of the nucleic acid, e.g., copies thatresult from amplification. Amplification may be desirable in certaininstances, e.g., in order to obtain a desired amount of material fordetecting variations. The amplicons may then be subjected to a variationdetection method, such as those described below, to determine expressionof certain genes.

Levels of mRNA may be measured and quantified by various methodswell-known to those skilled in the art, including use of commerciallyavailable kits and reagents. One such method is polymerase chainreaction (PCR). Another method, for quantitative use, is real-timequantitative PCR, or qPCR. See, e.g., “PCR Protocols, A Guide to Methodsand Applications,” (M. A. Innis et al., eds., Academic Press, Inc.,1990); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987, and periodic updates); and “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994).

A microarray is a multiplex technology that typically uses an arrayedseries of thousands of nucleic acid probes to hybridize with, e.g., acDNA or cRNA sample under high-stringency conditions. Probe-targethybridization is typically detected and quantified by detection offluorophore-, silver-, or chemiluminescence-labeled targets to determinerelative abundance of nucleic acid sequences in the target. In typicalmicroarrays, the probes are attached to a solid surface by a covalentbond to a chemical matrix (via epoxy-silane, amino-silane, lysine,polyacrylamide or others). The solid surface is for example, glass, asilicon chip, or microscopic beads. Various microarrays are commerciallyavailable, including those manufactured, for example, by Affymetrix,Inc. and Illumina, Inc.

A biological sample may be obtained using certain methods known to thoseskilled in the art. Biological samples may be obtained from vertebrateanimals, and in particular, mammals. In certain instances, a biologicalsample is synovial tissue, serum or peripheral blood mononuclear cells(PBMC). By screening such body samples, a simple early diagnosis can beachieved for diseases such as ulcerative colitis and Crohn's disease. Inaddition, the progress of therapy can be monitored more easily bytesting such body samples for variations in expression levels of targetnucleic acids (or encoded polypeptides).

Subsequent to the determination that a subject, or the tissue or cellsample comprises a gene expression signature or relative levels ofcertain biomarkers disclosed herein, it is contemplated that aneffective amount of an appropriate therapeutic agent may be administeredto the subject to treat the particular disease in the subject, e.g., UCor Crohn's disease. Clinical diagnosis in mammals of the variouspathological conditions described herein can be made by the skilledpractitioner. Clinical diagnostic techniques are available in the artwhich allow, e.g., for the diagnosis or detection of inflammatory boweldiseases in a mammal, e.g., ulcerative colitis and Crohn's disease.

Kits

For use in the applications described or suggested herein, kits orarticles of manufacture are also provided. Such kits may comprise acarrier means being compartmentalized to receive in close confinementone or more container means such as vials, tubes, and the like, each ofthe container means comprising one of the separate elements to be usedin the method. For example, one of the container means may comprise aprobe that is or can be detectably labeled. Such probe may be apolynucleotide specific for a polynucleotide comprising one or moregenes of a gene expression signature. Where the kit utilizes nucleicacid hybridization to detect the target nucleic acid, the kit may alsohave containers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label.

Kits will typically comprise the container described above and one ormore other containers comprising materials desirable from a commercialand user standpoint, including buffers, diluents, filters, needles,syringes, and package inserts with instructions for use. A label may bepresent on the container to indicate that the composition is used for aspecific therapy or non-therapeutic application, and may also indicatedirections for either in vivo or in vitro use, such as those describedabove. Other optional components in the kit include one or more buffers(e.g., block buffer, wash buffer, substrate buffer, and the like), otherreagents such as substrate (e.g., chromogen) which is chemically alteredby an enzymatic label, epitope retrieval solution, control samples(positive and/or negative controls), control slide(s) etc.

Methods of Marketing

The invention herein also encompasses a method for marketing atherapeutic agent or a pharmaceutically acceptable composition thereofcomprising promoting to, instructing, and/or specifying to a targetaudience, the use of the agent or pharmaceutical composition thereof fortreating a patient or patient population with a particular disease,e.g., UC or Crohn's disease, from which a sample has been obtainedshowing a gene expression signature or levels of serum biomarkers asdisclosed herein.

Marketing is generally paid communication through a non-personal mediumin which the sponsor is identified and the message is controlled.Marketing for purposes herein includes publicity, public relations,product placement, sponsorship, underwriting, and sales promotion. Thisterm also includes sponsored informational public notices appearing inany of the print communications media designed to appeal to a massaudience to persuade, inform, promote, motivate, or otherwise modifybehavior toward a favorable pattern of purchasing, supporting, orapproving the invention herein.

The marketing of the diagnostic method herein may be accomplished by anymeans. Examples of marketing media used to deliver these messagesinclude television, radio, movies, magazines, newspapers, the internet,and billboards, including commercials, which are messages appearing inthe broadcast media.

The type of marketing used will depend on many factors, for example, onthe nature of the target audience to be reached, e.g., hospitals,insurance companies, clinics, doctors, nurses, and patients, as well ascost considerations and the relevant jurisdictional laws and regulationsgoverning marketing of medicaments and diagnostics. The marketing may beindividualized or customized based on user characterizations defined byservice interaction and/or other data such as user demographics andgeographical location.

The foregoing written specification and following examples areconsidered to be sufficient to enable one skilled in the art to practicethe invention. Various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and following examples andfall within the scope of the appended claims.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein.

Further details of the invention are illustrated by the followingnon-limiting Examples. The disclosures of all citations in thespecification are expressly incorporated herein by reference.

EXAMPLES Example 1 Phase II Randomized Double-Blind Placebo-ControlledStudy to Evaluate the Efficacy and Safety of rhuMAb Beta7 (Etrolizumab)in Patients with Moderate to Severe Ulcerative Colitis and Open LabelExtension Study Description of the Clinical Study

Description of rhuMAb Beta7 (Etrolizumab)

RhuMAb Beta7 (etrolizumab) is a humanized monoclonal antibody based onthe human IgG1 subgroup III V_(II), κ subgroup-I V_(L) consensussequences and is directed specifically against the β7 subunit of theintegrin heterodimer. See FIGS. 1A and B. It has been shown to bind withhigh affinity to α4β7 (K_(d) of about 116 pM) and αEβ7 (K_(d) of about1800 pM).

This recombinant antibody has two heavy chains (446 residues) and twolight chains (214 residues) that are covalently linked by interchain andintrachain disulfide bonds typical of IgG1 antibodies. For the workdescribed herein, it was produced in Chinese hamster ovary (CHO) cells.The molecular mass of the intact, non glycosylated rhuMAb Beta7 moleculewas approximately 144 kDa. Each heavy chain of rhuMAb Beta7 has oneconserved N linked glycosylation site at Asn297. The oligosaccharidespresent at this site were typical of those observed in recombinantantibodies expressed in CHO cells, with the predominant glycoforms beingthe asialo, biantennary G0, and G1 glycans. The mass of the mostprevalent rhuMAb Beta7 form containing two G0 glycans and no C terminallysine residues was approximately 147 kDa.

RhuMAb Beta7 drug product and placebo were prepared by Genentech. Theywere clear to slightly opalescent, colorless to slightly yellow aqueoussolutions. Both solutions were sterile and preservative-free liquidintended for IV and SC administration.

Study Design Description of the Study

This Phase II study was a randomized, double-blind, placebo-controlledmulticenter study to evaluate the efficacy and safety across two rhuMAbBeta7 dose levels compared with placebo in patients with moderate tosevere UC. The primary efficacy endpoint was evaluated at Week 10 (2weeks after the final dose of study drug was administered) with asecondary efficacy endpoint at Week 6.

Patients were randomized in a 1:1:1 ratio across a dose range of rhuMAbBeta7 100 mg SC (flat dose) at Weeks 0, 4, and 8 and 420 mg SC (flatloading dose) at Week 0 followed by 300 mg SC at Weeks 2, 4, and 8 ormatching placebo SC. The study schema is shown in FIG. 2. The study wasdivided into a screening period of 0-35 days, a double-blind treatmentperiod of 10 weeks, a safety follow-up period of 18 weeks, and aprogressive multifocal leukoencephalopathy (PML) follow up period of 17months (2 years after randomization).

To be eligible, patients must have had a minimum of a 12-week durationof UC diagnosed according to the American College of Gastroenterology(ACG) practice guidelines; that is, clinical and endoscopic evidencecorroborated by a histopathology report, with evidence of moderate tosevere disease as evidenced by, in certain instances an MCS of ≥5, or incertain instances, an MCS of ≥6, including an endoscopy subscore of ≥2;a rectal bleeding subscore of ≥1 (see Table 1); and endoscopic evidenceof disease activity a minimum of 25 cm from the anal verge. Additionalinclusion and exclusion criteria for this study are provided in Intn'lPatent Pub. No. WO/2012/135589.

TABLE 1 Mayo Clinic Scoring System for Assessment of Ulcerative ColitisActivity. Assessment Category Physician's Stool Rectal Findings onglobal Score frequency^(a) bleeding^(b) Endoscopy assessment^(c) 0normal no. no blood normal or normal of stools seen inactive disease forthis patient 1 1 to 2 streaks of mild disease mild stools blood with(erythema, disease more than stool less decreased vascular normal thanhalf pattern, mild the time friability) 2 3 to 4 obvious moderatedisease moderate stools blood with (marked erythema, disease more thanstool most lack of vascular normal of the pattern, friability, timeerosions) 3 5 or more blood severe disease severe stools alone(spontaneous disease more than passes bleeding, normal ulceration)subscore: subscore: subscore: subscore: 0 to 3 0 to 3 0 to 3 0 to 3^(a)Each patient serves as his or her own control to establish thedegree of abnormality of the stool frequency. ^(b)The daily bleedingscore represents the most severe bleeding of the day. ^(c)Thephysician's global assessment acknowledges the three other criteria, thepatient's daily recollection of abdominal discomfort and general senseof well-being, and other observations, such as physical findings and thepatient's performance status.

Prior to randomization, patients must have been on stable doses ofconcomitant medications for UC. Oral 5-aminosalicylic acid (5-ASA) andimmunosuppressant (azathioprine [AZA], 6-mercaptopurine [6-MP], ormethotrexate) doses must have been kept stable for at least 4 weeksprior to randomization on Day 1. Patients who were receiving topical5-ASA or corticosteroids must have discontinued 2 weeks beforerandomization on Day 1. Oral corticosteroid doses must have been keptstable for 2 weeks prior to randomization on Day 1. Patients receivinghigh-dose steroids must have had the dose reduced to ≤20 mg/day for 2weeks prior to randomization on Day 1. For patients receiving oralcorticosteroids during the study treatment period, tapering of steroidsmust have been commenced at Week 10 at a rate of a 5-mg prednisone orprednisone equivalent per week for 2 weeks and then at a rate of 2.5 mgprednisone or prednisone equivalent per week to discontinuation. Forpatients receiving oral immunosuppressants (other than oralcorticosteroids), tapering of immunosuppressants must have beencommenced at Week 8, and patients must have completely discontinuedimmunosuppressants by Week 10. Patients who have previously receivedanti-TNF therapy must have discontinued therapy for a minimum of 8 weeksprior to randomization to receive study drug on Day 1. If patientsexperienced persisting or increasing disease activity at any time duringthe study, rescue therapy in the form of an increase in steroids and orimmunosuppressant dose may be increased or initiated according to theinvestigator's clinical judgment. Patients who required rescue therapywere permitted to remain in the study but discontinued study treatmentand, during data analysis, were classified as having experiencedtreatment failure.

Patients were assessed to determine whether they failed to respond toconventional therapy, including at least one anti-TNF agent. As usedherein, loss of response and/or intolerance to anti-TNF agents andimmunosuppressants means the following. With respect to anti-TNF agents,loss of response and/or intolerance means that symptoms of activedisease persist despite previous treatment with one or more of (a)infliximab: 5 mg/kg IV, 3 doses over 6 weeks with assessment at 8 weeks;(b) adalimumab: one 160-mg SC does at week 0, followed by one 80-mg doseat week 2 and then 40 mg at 4 and 6 weeks, with assessment at 8 weeks;or recurrent active symptoms during regularly scheduled maintenancedosing following a previous response (elective discontinuation oftreatment by patient who has responded and did not lose response doesnot qualify); or history of intolerance to at least one ant-TNF antibody(including but not exclusive of or limited to infusion-related reactionor injection-site reaction, infection, congestive heart failure,demyelination). With respect to immunosuppressant agents, loss ofresponse and/or intolerance means that symptoms of active diseasepersist despite previous treatment with one or more of azathioprine(≥1.5 mg/kg) or equivalent dose of 6-mercaptopurine mg/kg (≥0.75 mg/kg)or methotrexate, 25 mg SC/intramuscular (or as indicated) per week forat least 8 weeks; or history of intolerance of at least oneimmunosuppressive (including, but not exclusive of pancreatitis, drugfever, rash, nausea/vomiting, liver function test elevation, thiopurineS-methyltransferase genetic mutation, infection).

Randomization to study treatment were stratified by concomitanttreatment with corticosteroids (yes/no), concomitant treatment withimmunosuppressants (yes/no), previous anti-TNF exposure (yes/no) (exceptfor patients randomized in the United States), and study site.

UC disease activity was assessed using the MCS at Screening (and thiswas considered the baseline MCS), Week 6 (2 weeks after dosing at Week4), and Week 10 (2 weeks after the final dose of study drug). Biopsiesof the colon were obtained during the flexible sigmoidoscopy conductedat these same time points. Partial MCS was also collected throughout thestudy. Patient Reported Outcomes (PROs) were also assessed by using aShort Inflammatory Bowel Disease Questionnaire (SIBDQ) and MCS, whichwere to be completed by patients at Day 1 and at Weeks 6 and 10. Inaddition, disease activity, daily symptoms, and impact of UC werecollected in a patient diary, to be completed daily by patients fromscreening (approximately 7 days prior to and up to Day 1) and at least 7days prior to and up to the study visits at Weeks 6 and 10. Serum andfecal samples were also obtained for biomarker analysis. Stool wasobtained at screening and Weeks 6, 10, and 28 for measurement ofbiomarkers. Exemplary biomarkers that were considered for measurementinclude, but are not limited to, lipocalin, calprotectin, andlactorferrin. Serum and plasma were obtained at screening, at Day 1, andat Weeks 4, 6, 10, 16, and 28 for measurement of exploratory biomarkers.

The primary efficacy endpoint for this study was the proportion ofpatients who achieved clinical remission, defined as an absolutereduction in MCS to ≤2 with no individual subscore exceeding 1 point, byWeek 10. Additional secondary endpoints are listed in the study outcomemeasures as described below.

Outcome Measures Primary Efficacy Outcome Measure

The primary efficacy outcome measure was clinical remission at Week 10.Clinical remission is defined by an MCS ≤2 with no individual subscoreexceeding 1 point (see Table 1).

Secondary Efficacy Outcome Measures

The secondary efficacy outcome measures for this study were (1) Clinicalresponse at Week 6 and Week 10 where clinical response was defined by atleast a 3-point decrease and 30% reduction from baseline in MCS and a≥1-point decrease in rectal bleeding subscore or absolute rectalbleeding score of 0 or 1; (2) Clinical remission (defined above) at Week6; and (3) An indicator for endoscopic score and rectal bleeding scoreof 0 at Week 6 and Week 10.

Exploratory Outcome Measures

The exploratory outcome measure for this study were the time to flare ofUC in patients who achieved response or remission. For this outcomemeasure, a flare is defined as a 2 point increase in partial MCSaccompanied by 3 days of continuous rectal bleeding and an endoscopyscore of 2 on flexible sigmoidoscopy.

Safety Outcome Measures

The safety and tolerability of rhuMAb Beta7 were assessed using thefollowing measures: (1) Incidence of adverse events and serious adverseevents graded according to National Cancer Institute Common TerminologyCriteria for Adverse Events (NCI CTCAE) Version 4.0; (2) Clinicallysignificant changes in vital signs and safety laboratory measures; (3)Discontinuation due to adverse event(s); (4) Incidence and nature ofinjection-site reactions and hypersensitivity; (5) Incidence ofinfectious complications; and (6) Immunogenicity as measured by theincidence of ATAs.

Pharmacokinetic Outcome Measures

The pharmacokinetic outcome measures included the following: (1) C_(max)after the first and final doses; (2) Time to maximum concentration(T_(max)) after the first and final doses; (3) Area under the serumconcentration-time curve (AUC) within a dose interval after the finaldose; (4) AUC from time 0 to time of the last detectable observation(AUC_(last)) or to infinity (AUC_(inf)); (5) Apparent clearance (CL/F);(6) Apparent volume of distribution (V/F); and (7) Elimination half-life(t_(1/2)).

Example 2—Predictive Biomarker Studies and Analyses

Rationale for Selection of Predictive Biomarkers Integrin Beta7 andCD3epsilon for Enrichment of Efficacy in Patients Treated withEtrolizumab

As discussed above, etrolizumab binds to the 07 integrin and blocksbinding to MAdCAM1, which is expressed on mucosal endothelial cells.Binding of lymphocytes that express β7 integrin to MAdCAM1 plays animportant role in migration of cells to the small intestine, lymphoidfollicles of the large intestine, and mesenteric lymph nodes. Wehypothesized that because increased migration of gut homing lymphocytesvia α4β7:MAdCAM interactions is thought to drive on-going inflammationin UC, patients with evidence of such increased gut homing oflymphocytes would benefit from treatment with etrolizumab. We thusselected predictive diagnostic markers that measured lymphocyte and/orβ7:MAdCAM 1 pathway activity to look for increased efficacy in patientswho may have disease strongly driven by lymphocyte migration andpresence in disease tissue.

Candidate predictive diagnostic markers integrin beta7 and CD3epsiolonwere pre-selected prior to analysis of clinical efficacy results. Biopsyexpression of CD3ε, which is a component of the T cell receptor-CD3signaling complex, was chosen as a potential predictive biomarkerbecause of its relatively exclusive expression on T cells, which maydrive disease in the gut. In addition, β7 integrin is expressed onlymphocytes and dendritic cells and other cell types. As predicted bypreclinical and Phase I clinical studies, such β7-expressing cellsshould be blocked from trafficking to the gut in patients treated withetrolizumab. Accordingly, we sought to determine whether levels of β7integrin in biopsies and/or in peripheral blood were predictive forresponse to treatment with etrolizumab.

Collection and Processing of Intestinal Biopsy Tissue

Intestinal biopsies were collected from study participants duringflexible sigmoidoscopy/full colonoscopy at the screening visit (up to 4weeks prior to treatment). Biopsies were taken from the most inflamedarea of the colon within 10-40 cm of the anal verge. Biopsies withinnecrotic areas of ulcerated mucosa or at suture sites in patients withprior colonic resection were avoided. Biopsies were placed into a tissueRNA stabilizing buffer (RNAlater, Qiagen, Cat. No. 76104) and frozen forshipment. Upon receipt, the biopsy samples were thawed and homogenizedwith 3 mm steel beads using a TissueLyzer (Qiagen, Cat. No. 69989) andRNA was isolated using the RNeasy Mini kit (Qiagen, Cat. No. 74106)according to manufacturer's instructions. RNA integrity was assessedwith the Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Pico Kit(Agilent Technologies, Cat. No. 5067-1513). Samples with low RNA quality(RIN ≤5) were excluded from analysis. RNA was reverse transcribed intocDNA using the ABI high capacity RT kit (Life Technologies, Cat. No.4368814). Gene expression levels (i.e. RNA levels) were assessed byreal-time PCR, also referred to as quantitative PCR or qPCR. Real-timePCR reactions were run on the BioMark™ HD System with Fluidigm pre-ampmaster mix (Life Technologies, Cat. No. 4391128) and reagents (Fluidigm,Cat. No. BMK-M10-96.96) using human CD3ε (Cat. No. Hs00167894_m1), αE(Cat. No. Hs01025372_m1), (37 (Cat. No. Hs01565750_m1), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat. No.Hs99999905_m1) primer sets (all from Applied Biosystems [LifeTechnologies]) according to manufacturer's instructions. CD3ε, αE, andβ7 expression was normalized to GAPDH expression using thepreviously-described δCT method.

The results of these experiments are shown in FIGS. 3A-3C (β7 geneexpression) and 4A-C (CD3ε gene expression). In each bar graph plot, lowgene expression (below the median for the population of patients fromthe Phase II study) is shown on the left half of the plot for placebo,100 mg/dose etrolizumab, and 300 mg/dose etrolizumab and high geneexpression (above the median for the population of patients from thePhase II study) is shown on the right half of the plot for placebo, 100mg/dose etrolizumab, and 300 mg/dose etrolizumab. For both beta7 geneexpression and CD3ε expression at baseline (also referred to asscreening, i.e. prior to first drug or placebo administration), weassessed the percentage of patients who had achieved clinical response(FIG. 3C and FIG. 4C; defined as a 3-point decrease and 30% reductionfrom baseline in MCS and ≥1-point decrease in rectal bleeding subscoreor absolute rectal bleeding score of 0 or 1), mucosal healing (FIG. 3Band FIG. 4B; defined as an endoscopic subscore of 0 or 1) or remission(FIG. 3A and FIG. 4A; defined as MCS ≤2 with no individual subscore >1)at week 10 in the clinical study described above. All reported values(numbers above the bars) were corrected for placebo response in thebeta7 high and beta7 low populations (FIGS. 3A-3C) and CD3ε high andCD3ε low populations (FIGS. 4A-4C).

As can be seen in FIGS. 3A-3C, patients with high levels of beta7 geneexpression at baseline showed higher placebo-corrected response totreatment with 100 mg/dose etrolizumab as measured by all threeendpoints of (i) remission (31% beta7 high vs. 18% beta7 low), (ii)mucosal healing (37% beta7 high vs. 1% beta7 low), and (iii) clinicalresponse (29% beta7 high vs.−3% beta7 low). This enriched clinicalbenefit was not observed with the 300 mg/dose of etrolizumab for themucosal healing (14% beta7 high vs. 16% beta7 low) and remissionendpoints (7% beta7 high vs. 20% beta7 low), but was observed for theclinical response endpoint (21% beta7 high vs.−1% beta7 low). Similarly,patients with high levels of CD3ε gene expression at baseline showedhigher placebo-corrected response to treatment with 100 mg/doseetrolizumab (but not 300 mg/dose etrolizumab) as measured by all threeendpoints of clinical response (36% CD3ε high vs.−6% CD3ε low at 100mg/dose; 10% CD3ε high vs. 9% CD3ε low at 300 mg/dose), mucosal healing(36% CD3ε high vs. 5% CD3ε low at 100 mg/dose; 12% CD3ε high vs. 21%CD3ε low at 300 mg/dose), and remission (36% CD3ε high vs. 15% CD3ε lowat 100 mg/dose; 13% CD3ε high vs. 15% CD3ε low at 300 mg/dose).Accordingly, these results demonstrate that higher than median levels ofeither beta7 gene expression and/or CD3ε gene expression in intestinalbiopsies of inflamed areas of the colon are associated with increasedclinical benefit of etrolizumab treatment, and this clinical benefit isgreatest when etrolizumab is administered at 100 mg/dose. Thus, higherthan median levels of either beta7 gene expression and/or CD3ε geneexpression in intestinal biopsies of inflamed areas of the colon showpotential as predictive biomarkers to identify UC patients most likelyto benefit from treatment with therapeutic agents that target the beta7integrin subunit, including etrolizumab.

In certain instances, less invasive methods than obtaining intestinalbiopsies are desirable. Methods of assessing gene expression levels inperipheral blood are examples of such less invasive methods. Thus, wesought to determine whether the enriched clinical benefit seen inetrolizumab-treated patients who had higher than median levels ofbaseline beta7 gene expression in intestinal biopsy tissue could also beobserved by assessing beta7 gene expression levels in peripheral wholeblood samples.

In a Phase I trial, we had observed that patients who expressed highlevels of beta7 on CD3+ cells by FACS analysis appeared to respondbetter to etrolizumab treatment (data not shown) than patients who hadlow levels of beta7 on CD3+ cells. In that trial, we had not collectedperipheral blood for RNA analysis and thus could not analyze thosesamples for any correlations between beta7 RNA levels and beta7 proteinlevels and response to etrolizumab. Thus, prior to analyzing patientsamples from the Phase II trial, we sought to determine whether beta7gene expression levels (RNA levels) correlated with beta7 proteinexpression levels in CD3+ cells and in other blood cell populations insamples from IBD patients not enrolled in the Phase II trial and fromhealthy control subjects.

For the experiments described below, whole blood was collected accordingto standard procedures; blood for RNA studies was collected in PAXgenetubes according to the manufacturer's protocol. For FACS analysis,antibodies were obtained from Becton Dickenson (see Table 2). Cells fromperipheral blood samples were incubated with labeled antibodiesaccording to standard procedures known in the art and analyzed in aFACSCalibur machine. Beta7 expression was quantified using QuantumPE-MESF beads (Bangs Laboratory, Cat. No. 827). We also determined thatbeta7 expression by FACS analysis was optimum when patient samples wereshipped and/or stored overnight at room temperature prior to performingthe FACS analyses.

TABLE 2 Antibodies used for FACS analyses. Conjugate BD catalog# Beta7PE 555945 CD45RA FITC 347723 CD19 FITC 555412 CD27 APC 558664 CD3 APC340440 CD3 FITC 555332 CD3 PE 347347 CD4 PerCP Cy5.5 341654 IgD PerCPCy5.5 561315 mouse isotype IgG1 FITC 556649 mouse isotype IgG1 APC550854 mouse isotype IgG2a FITC 554647 mouse isotype IgG2a PerCP Cy5.5552577 mouse isotype IgG1 PerCP Cy5.5 550795 rat isotype IgG2a PE 555844

For qPCR, we generated cDNA using the BioRad iScript Select cDNASynthesis Kit (Cat. No. 170-8897) according to the manufacturer'sprotocol and then performed qPCR using ABI Taqman assays for integrinbeta7, CD3epsilon, CD20 (Cat. No. Hs00544819_m1), CD45 (Cat. No.Hs04189704_m1) and GAPDH according to the manufacturer's protocol. FIG.5 shows a plot correlating these FACS analysis and qPCR measurements. Ascan be seen in FIG. 5, beta7 RNA levels as measured by qPCR was notcorrelated with beta7 protein levels in CD3+ T cells or in CD19+ Bcells. However, beta7 protein levels in lymphocytes was significantlycorrelated with beta7 RNA levels normalized to the levels of thehousekeeping gene GAPDH or the RNA levels of CD45. See FIG. 5, boxedsquares, statistical correlations (Spearman r and p values indicated).Accordingly, we determined that beta7 gene expression (RNA levels) inperipheral blood (whole blood collected in PAXgene tubes) could bereliably detected and quantified by qPCR.

We next analyzed beta7 gene expression in the peripheral blood ofpatients treated with etrolizumab according to the Phase II protocoldescribed above and looked for associations between beta7 RNA levels inperipheral blood and efficacy measures.

Total RNA was isolated from frozen PAXgene blood tubes by automatedisolation on a KingFisher magnetic particle separator. Briefly, tubeswere allowed to thaw for 16 hours at room temperature. Aftercentrifugation and washing to collect white blood cell pellets, cellswere lysed in guanidinium-containing buffer. Organic extraction wasperformed prior to adding binding buffer and magnetic beads inpreparation for the KingFisher run. The procedure was optimized forretention of microRNAs and included a DNAse treatment step and cleanupprior to elution from the magnetic beads. The purity and quantity oftotal RNA samples were determined by absorbance readings at 260 and 280nm using a NanoDrop ND-1000 UV spectrophotometer. The integrity of totalRNA was qualified by Agilent Bioanalyzer 2100 microfluidicelectrophoresis, using the Nano Assay and the Caliper LabChip system.

Total RNA (up to 2 μg) was reverse transcribed reverse transcribed forone hour in a total reaction volume of 20 uL using the TaqMan® HighCapacity cDNA Synthesis Kit (Applied Biosystems [Life Technologies])according to the manufacturer's instructions. Fast real-time PCRreactions were run on a Viia™ 7 Real-Time PCR System (Applied Biosystems[Life Technologies]) using human 37 and GAPDH primer sets (AppliedBiosystems [Life Technologies]) according to manufacturer'sinstructions. Beta7 expression was normalized to GAPDH expression.

The results are shown in FIGS. 6A-6C. Patients with high levels of beta7peripheral blood gene expression at baseline showed greaterplacebo-corrected response to treatment with 100 mg/dose etrolizumab forthe mucosal healing endpoint (30% beta7 high vs. 3% beta7 low) (FIG. 6B)and the remission endpoint (42% beta7 high vs. 11% beta7 low) (FIG. 6A).Patients expressing high beta7 in the 100 mg/dose etrolizumab arm alsoshowed a higher response to etrolizumab treatment than patientsexpressing low levels of beta7 for the clinical response endpoint, butthese results were not placebo-corrected (FIG. 6C). The enrichedclinical benefit seen in beta7 high patients in the 100 mg/dose arm wasnot observed with the 300 mg/dose of etrolizumab for the mucosal healing(FIG. 6B) and clinical response endpoints (FIG. 6C), but was observedfor the remission endpoint (FIG. 6A), although the magnitude of thedifference between beta7 high patients and beta7 low patients was low.Accordingly, these results demonstrate that higher than median levels ofbeta7 gene expression in peripheral blood are associated with increasedclinical benefit of etrolizumab treatment, and this clinical benefit isgreatest when etrolizumab is administered at 100 mg/dose. Thus, higherthan median levels of beta7 gene expression in peripheral blood showspotential as a predictive biomarker to identify IBD patients, such as UCand Crohn's disease patients, most likely to benefit from treatment withtherapeutic agents that target the beta7 integrin subunit, includingetrolizumab.

Rationale for Selection of Predictive Biomarker Integrin alphaE forEnrichment of Efficacy in Patients Treated with Etrolizumab

Etrolizumab binds to the β7 integrin and blocks binding to both MAdCAM1,which is expressed on mucosal endothelial cells, and E-cadherin, whichis expressed on epithelial cells. Binding of lymphocytes that expressαEβ7 integrin to E-cadherin may help position and retain cells adjacentto the intestinal epithelium. We hypothesized that binding ofαEβ7:E-cadherin may also be important in on-going inflammation in UC andtherefore patients with evidence of increased αEβ7+ lymphocytes wouldbenefit from treatment with etrolizumab. We thus expanded our predictivediagnostic markers to include αE in our β7 pathway markers to look forincreased efficacy in patients who may have disease strongly driven bylymphocyte migration and presence in disease tissue. Accordingly, wesought to determine whether levels of αE integrin in biopsies and/or inperipheral blood were predictive for response to treatment withetrolizumab.

Intestinal biopsies were collected, placed in RNAlater and processed asdescribed above. Additional biopsies were taken and placed into formalinand stored until shipment. Upon receipt, the second set of biopsysamples were embedded in paraffin blocks. For the studies described inthis section, intestinal biopsies were either collected during the PhaseII trial described above or were obtained from other sources wheresamples were collected from patients undergoing intestinal biopsyprocedures for reasons unrelated to the above-described Phase 11 trial.Paraffin blocks were processed into 4 μm sections and placed on glassslides. Staining was performed on a Ventana Benchmark XT (VentanaMedical Systems; Tucson, Ariz.) autostainer. Pretreatment was done withCell Conditioner 1 for the recommended time. Primary antibody, anti-αE(rabbit monoclonal EPR4166 (2), Cat. No. ab129202, Abcam, Cambridge,Mass.), was used at a concentration of 1.896 μg/ml and was incubated onslides for 60 minutes at 37° C. This step was followed by incubation ofslides with Ventana Ultraview (Ventana Medical Systems; AZ). Ventana DABand Hematoxylin II were used for chromogenic detection and counterstain.

As shown in FIG. 7A, immunohistochemistry for αE demonstrated stainingof small round cells likely to be lymphocytes. A majority of these cellswere adjacent to or associated with epithelial cell crypts in the colon(FIG. 7A, left panels) and within villi in the small intestine (FIG. 7A,right panels). We also observed association with surface epithelium(data not shown). A subpopulation of larger cells with more diffusestaining was also observed and could represent dendritic cells. Weenumerated αE+ cells in stained sections from a panel of colon, ileumand jejunum tissue samples from non-IBD patients. Computerizedautocounts were used to enumerate αE+ cells on stained slides; analysiswas limited to cells that displayed radial symmetry in the mucosal areaof the tissue section. Increased numbers of αE+ cells per total cellswere observed in jejunum and ileum in comparison to colonic tissuesamples (FIG. 7B). Increased numbers of αE+ cells as a percentage oftotal cells was also observed in ileum and jejunum samples from CDpatients (FIG. 7B). Finally, we observed no difference between αE+ cellsas a percentage of total cells in tissue samples from IBD patients incomparison to non-IBD patients. Gene expression of αE normalized toGAPDH was also performed in samples from non-IBD and UC colon, and CDcolon, ileum and jejunum (FIG. 7C). Again, increased αE gene expressionwas observed in the small bowel and no differences were observed betweenIBD and non-IBD tissue samples.

The small bowel is of particular interest in CD, as a majority (50-60%)of CD patients have disease that includes the terminal ileum. The higherlevels of both αE+ cells and αE gene expression in small bowel samplesmay indicate that these cells play a key role in CD pathobiology andtheir relative abundance may thus serve as a predictive marker in CDpatients for treatment with etrolizumab. AlphaE+ cells, along withalphaE gene expression, in screening biopsy samples were considered aspotential predictive biomarkers in the Phase II study of etrolizumab inUC (described above).

Formalin fixed screening tissue biopsies embedded in paraffin blocksfrom patients enrolled in the Phase II study were sectioned and stainedfor αE by immunohistochemistry and counted as detailed above. Screeningbiopsies on enrolled patients with available tissue samples were used toestablish a median αE+ cells per total cell cutoff (FIG. 7D (wherestippled dots represent screening levels in patients treated withetrolizumab who went on to remission, open dots represent patients whodid not achieve remission, and black dots represent patients whoreceived placebo; dotted line indicates median; TNF naïve and TNFinadequate responders [TNF-IR] are also shown). FIG. 7E shows examplesof αE staining in screening biopsies from patients enrolled in the PhaseII study that had levels of αE+ cells per total cells that were above(αE high) and below (αE low) the median. The distribution of αE geneexpression in screening biopsies from patients enrolled in the Phase IIstudy is shown in FIG. 7F and the relationship of the two measures isshown in FIG. 7G.

The performance of αE as determined by gene expression andimmunohistochemistry using intestinal biopsies from patients are shownin FIGS. 8A-8C and 8D-F, respectively. In these experiments, patientswere not stratified by TNF status (TNF-naïve or TNF-IR). In each bargraph plot, low gene expression or αE+ cell numbers (below the medianfor the population of patients from the Phase II study) is shown on theleft half of the plot for placebo, 100 mg/dose etrolizumab, and 300mg/dose etrolizumab and high gene expression (above the median for thepopulation of patients from the Phase II study) is shown on the righthalf of the plot for placebo, 100 mg/dose etrolizumab, and 300 mg/doseetrolizumab. Data from all patients is shown. For both αE geneexpression and immunohistochemistry at baseline (also referred to asscreening, i.e. prior to first drug or placebo administration), weassessed the percentage of patients who had achieved clinical response(FIG. 8C and FIG. 8F; defined as a 3-point decrease and 30% reductionfrom baseline in MCS and >1-point decrease in rectal bleeding sub scoreor absolute rectal bleeding score of 0 or 1), mucosal healing (FIG. 8Band FIG. 8E; defined as an endoscopic subscore of 0 or 1) or remission(FIG. 8A and FIG. 8D; defined as MCS <2 with no individual subscore >1)at week 10 in the clinical study described above. All reported values(numbers above the bars) represent number of patients that achieved thisendpoint (numerator) over the number of patients in each dose group thatwere part of the analysis (denominator).

As can be seen in FIGS. 8A-8C, patients with high levels of αE geneexpression at screening showed higher placebo-corrected response totreatment with 100 mg/dose etrolizumab as measured by mucosal healing(19% αE high vs. 13% αE low), and remission (38% αE high vs. 13% αElow). This enriched clinical benefit was not observed with the 300mg/dose of etrolizumab. Patients with high levels of αE+ cells per totalcells showed higher placebo-corrected response to treatment with 100mg/dose etrolizumab as measured by mucosal healing (FIG. 8E; 19% αE highvs.−3% αE low), and remission (FIG. 8D; 50% αE high vs. 7% αE low). Inthis experiment, only remission was enriched in patients given 300mg/dose with high levels of αE+ cells per total cells at screening (FIG.8D; 14% αE high vs. 9% αE low).

FIGS. 9A-9F show results in TNF naïve patients. TNF naïve patients withhigh levels of αE gene expression at screening showed higherplacebo-corrected response to treatment with 100 mg/dose etrolizumab asmeasured by all three endpoints of clinical response (28% αE high vs.13% αE), mucosal healing (42% αE high vs. 17% αE), and remission (67% αEhigh vs. 17% αE low); only remission was enriched in patients given 300mg/dose etrolizumab (50% αE high vs. 20% αE low at 300 mg/dose) (FIGS.9A-9C). Likewise, TNF naïve patients with high levels of αE+ cells pertotal cells at screening showed higher placebo-corrected response totreatment with etrolizumab as measured by mucosal healing (FIG. 9E; 38%αE high vs. 25% αE in 100 mg/dose group and 46% αE high vs. 25% αE in300 mg/dose group) and remission (FIG. 9D; 67% αE high vs. 25% αE in 100mg/dose group and 50% αE high vs. 25% αE in 300 mg/dose group).

As discussed previously, less invasive methods than obtaining intestinalbiopsies are desirable. Methods of assessing gene expression levels inperipheral blood are examples of such less invasive methods. Since wehad already examined enriched clinical benefit in etrolizumab-treatedpatients by assessing beta7 gene expression levels in peripheral wholeblood samples and beta7 can pair with both α4 and αE, we next usedscreening and day 1 peripheral blood gene expression of αE to test forenrichment of response and remission. The results of these experimentsare shown in FIGS. 10A-10F. In these experiments, patients were notstratified by TNF status (TNF-naïve or TNF-IR).

As shown in FIG. 10A, patients with higher levels of αE gene expressionin peripheral blood at screening showed greater placebo-correctedresponse to treatment with etrolizumab for the remission endpoint (FIG.10A; 29% αE high vs. 8% αE low in 100 mg/dose group, 19% αE high vs. 5%αE low in 300 mg/dose group). AlphaE gene expression was also measuredin day 1 peripheral blood and αE high patients in the 100 mg/dose groupwere found to have increased remission (FIG. 10D; 33% αE high vs. 13% αElow) and mucosal healing (FIG. 10E; 27% αE high vs. 2% αE low).

FIGS. 11A-11F shows peripheral blood gene expression results atscreening or at day 1 in TNF naïve patients. TNF naïve patients withhigh levels of peripheral blood αE gene expression showed higherplacebo-corrected remission to treatment with etrolizumab at screening(FIG. 11A; 29% αE high vs. 8% αE in 100 mg/dose group and 40% αE highvs. 17% αE in 300 mg/dose group) and day 1 (FIG. 11D; 55% αE high vs.20% αE).

Patients enrolled in the Phase 11 study were given the option ofenrolling in an open-label extension study in which all patientsreceived 300 mg/dose of etrolizumab every four weeks as described above.Patients that were not in response could enter the study followingcompletion of the 10 week timepoint or at any time during safetyfollow-up. As shown in FIG. 12, 33 TNF-IR patients (14 in the 100mg/dose arm and 19 in the 300 mg/dose arm) that received active drug inthe Phase II study but did not have a response at 10 weeks enrolled inthe open-label extension study without interruption of the dosingregimen. These patients were scored for remission after four weeks(14-16 weeks after first dose) using the partial Mayo clinic score. Thepartial Mayo clinic score is a 9 point clinical score in which all Mayosubscores are collected except the endoscopic subscore. Remission isdefined as a total score of ≤2 points and response is defined as areduction in baseline partial Mayo clinic score by 25% and ≥2 points.

TNF-IR patients with high levels of αE gene expression in intestinalbiopsies at screening that did not have a response at the 10 weekprimary endpoint showed higher remission (FIG. 13A; 18% αE high vs. 0%αE low) and response (FIG. 13B; 53% αE high vs. 8% αE low) to continuedtreatment. TNF-IR patients with high levels of αE+ cells per total cellsat screening also showed higher remission (FIG. 13C; 22% αE high vs. 0%αE low) and response (FIG. 13D; 56% αE high vs. 17% αE low) toadditional treatment. TNF-IR patients with high levels of αE inperipheral blood showed higher response (FIG. 13F; 41% QE high vs. 15%αE low).

Accordingly, these results demonstrate that higher than median levels ofαE gene expression or αE+ cells per total cells in intestinal biopsiesof inflamed areas of the colon as well as higher than median levels ofαE gene expression in peripheral blood were associated with increasedclinical benefit of etrolizumab treatment in patients, and this clinicalbenefit was greatest when etrolizumab was administered at 100 mg/dosesubcutaneously every four weeks. Thus, higher than median levels of αEgene expression or αE+ cells in intestinal biopsies of inflamed areas ofthe colon or QE gene expression in peripheral blood show potential aspredictive biomarkers to identify IBD patients, such as UC and Crohn'sdisease patients, most likely to benefit from treatment with therapeuticagents that target the beta7 integrin subunit, including etrolizumab.

1.-24. (canceled)
 25. A method of predicting the responsiveness of apatient suffering from a gastrointestinal inflammatory disorder to atherapy comprising an integrin beta7 antagonist, the method comprising:obtaining a biological sample from the patient, measuring the number ofcells expressing at least one protein selected from the group consistingof integrin beta7, integrin alphaE, and CD3epsilon, comparing the numberof expressing cells measured in the sample to a reference level, andpredicting that the patient will likely respond to the therapy when thenumber of protein-expressing cells has been measured as elevatedcompared to the reference level and predicting that the patient will notlikely respond to the therapy when the number of protein-expressingcells has been measured as low compared to the reference level. 26.(canceled)
 27. A method of identifying a patient suffering from agastrointestinal inflammatory disorder as likely to respond to a therapycomprising an integrin beta7 antagonist, the method comprising: (a)measuring the number of cells expressing at least one protein in abiological sample from the patient, wherein the at least one protein isselected from the group consisting of integrin beta7, integrin alphaE,and CD3epsilon; (b) comparing the number of cells measured in (a) to areference level; and (c) identifying the patient as more likely torespond to the therapy comprising an integrin beta7 antagonist when thenumber of cells measured in (a) is above the reference level.
 28. Amethod of treating a patient having a gastrointestinal inflammatorydisorder, the method comprising: (a) measuring the number of cellsexpressing at least one protein in a biological sample from the patient,wherein the at least one protein is selected from the group consistingof integrin beta7, integrin alphaE, and CD3epsilon; (b) comparing thenumber of expressing cells measured in (a) to a reference level; (c)identifying the patient as more likely to respond a therapy comprisingan integrin beta7 antagonist when the number of protein-expressing cellsmeasured in (a) is above the reference level; and (d) administering thetherapy when the number of protein-expressing cells measured in (a) isabove the reference level, thereby treating the gastrointestinalinflammatory disorder.
 29. The method of claim 28, wherein about 100 mgof the integrin beta7 antagonist is administered subcutaneously onceevery four weeks.
 30. The method of claim 28, wherein the patient is ahuman.
 31. The method of claim 28, wherein the patient is a TNFinadequate responder (TNF-IR).
 32. The method of claim 28, wherein thegastrointestinal inflammatory disorder is an inflammatory bowel disease.33. The method of claim 32, wherein the inflammatory bowel disease isulcerative colitis or Crohn's disease.
 34. The method of claim 33,wherein the inflammatory bowel disease is ulcerative colitis and theresponse is selected from clinical response, mucosal healing andremission.
 35. The method of claim 28, wherein the biological sample isintestinal tissue.
 36. The method of claim 28, comprising measuring thenumber of expressing cells by an immunohistochemical method.
 37. Themethod of claim 28, wherein the measuring comprises contacting thesample with an agent that specifically binds to integrin beta7 protein,integrin alphaE protein, or CD3epsilon protein and detecting the amountof complex formed, and thereby measuring the number ofprotein-expressing cells.
 38. The method of claim 28, wherein the numberof expressing cells in the sample is divided by the total number ofcells in the sample.
 39. The method of claim 28, wherein the number ofexpressing cells in a high power field under a light microscope isdetermined.
 40. The method of claim 28, wherein the reference level is amedian value.
 41. (canceled)
 42. The method of claim 28, wherein theintegrin beta7 antagonist is a monoclonal anti-beta7 antibody or anantigen-binding fragment thereof.
 43. The method of claim 42, whereinthe anti-beta7 antibody is selected from the group consisting of achimeric antibody, a human antibody, and a humanized antibody. 44.(canceled)
 45. The method of claim 42, wherein the anti-beta7 antibodyor antigen-binding fragment thereof comprises six hypervariable regions(HVRs), wherein: (i) the HVR-L1 comprises amino acid sequence A1-A11,wherein A1-A11 is RASESVDTYLH (SEQ ID NO: 1); RASESVDTLLH (SEQ ID NO:7),RASESVDTLLH (SEQ ID NO:8), or RASESVDDLLH (SEQ ID NO:9) or a variant ofSEQ ID NOs:1, 7, 8 or 9 (SEQ ID NO:26) wherein amino acid A2 is selectedfrom the group consisting of A, G, S, T, and V and/or amino acid A3 isselected from the group consisting of S, G, I, K, N, P, Q, R, and T,and/or A4 is selected from the group consisting of E, V, Q, A, D, G, H,I, K, L, N, and R, and/or amino acid A5 is selected from the groupconsisting of S, Y, A, D, G, H, I, K, N, P, R, T, and V, and/or aminoacid A6 is selected from the group consisting of V, R, I, A, G, K, L, M,and Q, and/or amino acid A7 is selected from the group consisting of D,V, S, A, E, G, H, I, K, L, N, P, S, and T, and/or amino acid A8 isselected from the group consisting of D, G, N, E, T, P and S, and/oramino acid A9 is selected from the group consisting of L, Y, I and M,and/or amino acid A10 is selected from the group consisting of L, A, I,M, and V and/or amino acid A11 is selected from the group consisting ofH, Y, F, and S; (ii) the HVR-L2 comprises amino acid sequence B1-B8,wherein B1-B8 is KYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID NO:20), orXaaYASQSIS (SEQ ID NO:21, where Xaa represents any amino acid) or avariant of SEQ ID NOs:2, 20 or 21 (SEQ ID NO:27) wherein amino acid B1is selected from the group consisting of K, R, N, V, A, F, Q, H, P, I,L, Y and Xaa (where Xaa represents any amino acid), and/or amino acid B4is selected from the group consisting of S and D, and/or amino acid B5is selected from the group consisting of Q and S, and/or amino acid B6is selected from the group consisting of S, D, L, and R, and/or aminoacid B7 is selected from the group consisting of I, V, E, and K; (iii)the HVR-L3 comprises amino acid sequence C1-C9, wherein C1-C9 isQQGNSLPNT (SEQ ID NO:3) or a variant of SEQ ID NO:3 (SEQ ID NO:28)wherein amino acid C8 is selected from the group consisting of N, V, W,Y, R, S, T, A, F, H, I L, and M; (iv) the HVR-H1 comprises amino acidsequence D1-D10 wherein D1-D10 is GFFITNNYWG (SEQ ID NO:4); (v) theHVR-H2 comprises amino acid sequence E1-E17 wherein E1-E17 isGYISYSGSTSYNPSLKS (SEQ ID NO:5), or a variant of SEQ ID NO:5 (SEQ IDNO:29) wherein amino acid E2 is selected from the group consisting of Y,F, V, and D, and/or amino acid E6 is selected from the group consistingof S and G, and/or amino acid E10 is selected from the group consistingof S and Y, and/or amino acid E12 is selected from the group consistingof N, T, A, and D, and/or amino acid E13 is selected from the groupconsisting of P, H, D, and A, and/or amino acid E15 is selected from thegroup consisting of L and V, and/or amino acid E17 is selected from thegroup consisting of S and G; and (vi) the HVR-H3 comprises amino acidsequence F2-F11 wherein F2-F11 is MTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF(SEQ ID NO: 19); or comprises amino acid sequence F1-F11, wherein F1-F11is AMTGSSGYFDF (SEQ ID NO: 16), ARTGSSGYFDF (SEQ ID NO: 17), orAQTGSSGYFDF (SEQ ID NO: 18), or a variant of SEQ ID NOs:6, 16, 17, 18,or 19 (SEQ ID NO:30) wherein amino acid F2 is R, M, A, E, G, Q, S,and/or amino acid Flt is selected from the group consisting of F and Y.46. The method of claim 45, wherein the anti-beta7 antibody orantigen-binding fragment thereof comprises three heavy chainhypervariable region (HVR-H1-H3) sequences and three light chainhypervariable region (HVR-L1-L3) sequences, wherein: (i) the HVR-L1comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8or SEQ ID NO:9; (ii) the HVR-L2 comprises the amino acid sequence setforth in SEQ ID NO:2; HVR-L3 comprises SEQ ID NO:3; (iv) the HVR-H1comprises the amino acid sequence set forth in SEQ ID NO:4; (v) theHVR-H2 comprises the amino acid sequence set forth in SEQ ID NO:5; and(vi) the HVR-H3 comprises the amino acid sequence set forth in SEQ IDNO:6 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO:
 19. 47. The methodof claim 46, wherein the anti-beta7 antibody or antigen-binding fragmentthereof comprises a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:24 and a heavy chain variableregion comprising the amino acid sequence set forth in SEQ ID NO:31. 48.The method of claim 47, wherein the anti-beta7 antibody is etrolizumab.49.-50. (canceled)